Novel polymers and dna copolymer coatings

ABSTRACT

Some embodiments described herein relate to new polymer coatings for surface functionalization and new processes for grafting pre-grafted DNA-copolymers to surface(s) of substrates for use in DNA sequencing and other diagnostic applications.

INCORPORATION BY REFERENCE TO PRIORITY APPLICATIONS

The present application is a divisional of U.S. patent application Ser.No. 15/809,656, filed Nov. 10, 2017, to be issued as U.S. Pat. No.10,208,142 on Feb. 19, 2019, which is a continuation of U.S. patentapplication Ser. No. 14/927,252, filed Oct. 29, 2015, which claims thebenefit of priority to U.S. Provisional Patent Application No.62/073,764, filed on Oct. 31, 2014, each of which is hereby expresslyincorporated by reference in its entirety.

REFERENCE TO SEQUENCE LISTING

The present application includes a sequence listing in Electronicformat. The Sequence Listing is provided as a file entitledILLINC267D1_sequence_listing.txt, which is approximately 1 KB in size.The information in the electronic format of the sequence listing isincorporated herein by reference in its entirety.

FIELD

In general, the present application relates to the fields of chemistry,biology and material science. More specifically, the present applicationrelates to novel polymer coatings and grafted DNA-copolymers to supportsubstrate surface functionalization and downstream applications, such asDNA sequencing and other diagnostic applications. Methods for preparingsuch functionalized surface and the use thereof are also disclosed.

BACKGROUND

Polymer or hydrogel-coated substrates are used in many technologicalapplications. For example, implantable medical devices can be coatedwith biologically inert polymers. In another example, polymer orhydrogel coated substrates are used for the preparation and/or analysisof biological molecules. Molecular analyses, such as certain nucleicacid sequencing methods, rely on the attachment of nucleic acid strandsto a polymer or hydrogel-coated surface of a substrate. The sequences ofthe attached nucleic acid strands can then be determined by a number ofdifferent methods that are well known in the art.

In certain Sequencing-by-Synthesis (“SBS”) processes, one or moresurfaces of a flow cell are coated with a polymer or a hydrogel to whichprimers (single stranded DNA or ssDNA) are then grafted. However, thereis an inherent cost associated with performing the coating, grafting andquality control steps.

SUMMARY

The present application discloses polymer coatings that are useful forSBS applications and processes of incorporating the primer polymercoupling steps into the initial polymer synthesis. This may eliminatesome or all of the grafting process undertaken to manufacture asequencing flow cell or other substrate used for SBS. These processesmay maximize primer accessibility to the downstream biochemistry,minimize side reactions and yield a more efficient surface chemistry.The coatings and processes disclosed herein are useful for otheranalytical apparatus and processes including, but not limited to, thoseused for synthesis or detection of nucleic acids and other biologicallyactive molecules.

Some embodiments described herein are related to a polymer for surfacefunctionalization, comprising a recurring unit of Formula (I) and arecurring unit of Formula (II):

wherein each R^(1a), R^(2a), R^(1b) and R^(2b) is independently selectedfrom hydrogen, optionally substituted alkyl or optionally substitutedphenyl; each R^(3a) and R^(3b) is independently selected from hydrogen,optionally substituted alkyl, optionally substituted phenyl, oroptionally substituted C₇₋₁₄ aralkyl; and each L¹ and L² isindependently selected from an optionally substituted alkylene linker oran optionally substituted heteroalkylene linker. In some embodiments,the polymer may further comprise one or more recurring units selectedfrom the group consisting of polyacrylamides, polyacrylates,polyurethanes, polysiloxanes, silicones, polyacroleins,polyphosphazenes, polyisocyanates, poly-ols, and polysaccharides, orcombinations thereof. In some such embodiments, the polymer may furthercomprise one or more recurring units of polyacrylamide of Formula (IIIa)or (IIIb) or both:

wherein each R^(4a), R^(4b) and R^(5b) is selected from hydrogen or C₁₋₃alkyl; and each R^(5a), R^(6a), R^(6b), and R^(7b) is independentlyselected from hydrogen, optionally substituted C₁₋₆ alkyl or optionallysubstituted phenyl.

Some embodiments described herein are related to a substrate having afirst surface comprising a polymer with a recurring unit of Formula (I)and a recurring unit of Formula (II) as described herein. In someembodiments, the polymer may further comprise one or more recurringunits of various different polymer backbones as described above, forexample, one or more recurring units of polyacrylamide of Formula (IIIa)or (IIIb) or both.

In some embodiments, when the polymer is covalently attached to thefirst surface of the substrate, at least one covalent bond is formedbetween the amino group of the recurring unit of Formula (II) and thefirst surface of the substrate. Therefore, as described herein, asubstrate having a first surface comprising a polymer with a recurringunit of Formula (I) and a recurring unit of Formula (II) covalentlybonded thereto, should be understood to also include the polymer with amodified recurring unit of the structure

showing the covalent bonding position with the substrate surface.

Some embodiments described herein are related to a grafted polymercomprising functionalized oligonucleotides covalently bonded to apolymer with a recurring unit of Formula (I) and a recurring unit ofFormula (II) as described herein. In some embodiments, the polymer mayfurther comprise one or more recurring units of various differentpolymer backbones as described above, for example, one or more recurringunits of polyacrylamide of Formula (IIIa) or (IIIb) or both.

In some embodiments, when functionalized oligonucleotides are covalentlybonded to the polymer, at least two covalent bonds are formed as theresult of a reaction between the azido group of the recurring unit ofFormula (I) and a functionalized oligonucleotide. Therefore, asdescribed herein, a grafted polymer comprising functionalizedoligonucleotides covalently bonded to a polymer of a recurring unit ofFormula (I) and a recurring unit of Formula (II), should be understoodto also include the polymer with a modified recurring unit of thestructure

showing the covalent bonding position with functionalizedoligonucleotide, wherein

is a single or double bond.

Some embodiments described herein are related to a polymer for surfacefunctionalization, comprising a recurring unit of Formula (IV):

wherein each R^(1c) and R^(2c) is independently selected from hydrogen,optionally substituted alkyl or optionally substituted phenyl; R^(3c) isselected from hydrogen, optionally substituted alkyl, optionallysubstituted phenyl, or optionally substituted C₇₋₁₄ aralkyl; Ar isselected from an optionally substituted C₆₋₁₀ aryl or an optionallysubstituted 5 or 6 membered heteroaryl; R^(A) is optionally substitutedtetrazine; and L³ is selected from a single bond, an optionallysubstituted alkylene linker or an optionally substituted heteroalkylenelinker. In some embodiments, the polymer may further comprise one ormore recurring units selected from the group consisting ofpolyacrylamides, polyacrylates, polyurethanes, polysiloxanes, silicones,polyacroleins, polyphosphazenes, polyisocyanates, poly-ols, andpolysaccharides, or combinations thereof. In some such embodiments, thepolymer may further comprise one or more recurring units ofpolyacrylamide of Formula (IIIa) or (IIIb) or both, with the structureshown above.

Some embodiments described herein are related to a substrate having afirst surface comprising a polymer with a recurring unit of Formula (IV)as described herein. In some embodiments, the polymer may furthercomprise one or more recurring units of various different polymerbackbones as described above, for example, one or more recurring unitsof polyacrylamide of Formula (IIIa) or (IIIb) or both.

In some embodiments, when the polymer is covalently attached to thefirst surface of the substrate, at least two covalent bonds are formedas the result of a reaction between the tetrazine group of the recurringunit of Formula (IV) and the first surface of the substrate. In someother embodiments, at least two covalent bonds are formed between thetetrazine group of the recurring unit of Formula (IV). Therefore, asdescribed herein, a substrate having a first surface comprising apolymer with a recurring unit of Formula (IV) as described herein,should be understood to also include the polymer with a modifiedrecurring unit of the structure

wherein the moiety Ar—R^(AA) is selected from

showing the covalent bonding position with the substrate surface; andwherein

is a single or double bond. R^(AA) may be optionally substituted.

Some embodiments described herein are related to a grafted polymercomprising functionalized oligonucleotides covalently bonded to apolymer with a recurring unit of Formula (IV) as described herein. Insome embodiments, the polymer may further comprise one or more recurringunits of various different polymer backbones as described above, forexample, one or more recurring units of polyacrylamide of Formula (IIIa)or (IIIb) or both.

In some embodiments, when functionalized oligonucleotides are covalentlybonded to the polymer, at least two covalent bonds are formed as theresult of a reaction between the tetrazine group of the recurring unitof Formula (IV) and a functionalized oligonucleotide. Therefore, asdescribed herein, a grafted polymer comprising functionalizedoligonucleotides covalently bonded to a polymer of a recurring unit ofFormula (IV), should be understood to also include the polymer with amodified recurring unit of the structure

wherein the moiety Ar—R^(AB) is selected from

showing the covalent bonding position with the oligonucleotide; andwherein

is a single or double bond. R^(AB) may be optionally substituted.

Some embodiments described herein are related to a polymer for surfacefunctionalization, comprising a recurring unit of Formula (V):

wherein each R^(1d) and R^(2d) is independently selected from hydrogen,optionally substituted alkyl or optionally substituted phenyl; eachR^(3d) is selected from hydrogen, optionally substituted alkyl,optionally substituted phenyl, or optionally substituted C₇₋₁₄ aralkyl;R^(B) is selected from azido, optionally substituted amino,Boc-protected amino, hydroxy, thiol, alkynyl, alkenyl, halo, epoxy,tetrazinyl or aldehyde; each L⁴ and L⁵ is independently selected from anoptionally substituted alkylene linker or an optionally substitutedheteroalkylene linker. In some embodiments, the polymer may furthercomprise a recurring unit of Formula (VIa) or (VIb), or both:

wherein each R^(1e), R^(2e), R^(1f) and R^(2f) is independently selectedfrom hydrogen, optionally substituted alkyl or optionally substitutedphenyl. In some embodiments, the polymer may further comprise one ormore recurring units selected from the group consisting ofpolyacrylamides, polyacrylates, polyurethanes, polysiloxanes, silicones,polyacroleins, polyphosphazenes, polyisocyanates, poly-ols, andpolysaccharides, or combinations thereof.

Some embodiments described herein are related to a substrate having afirst surface comprising a polymer with a recurring unit of Formula (V)as described herein. In some embodiments, the polymer may furthercomprise a recurring unit of Formula (VIa) or (VIb), or both. In someembodiments, the polymer may further comprise one or more recurringunits of various different polymer backbones as described above.

In some embodiments, when the polymer is covalently attached to thefirst surface of the substrate, at least two covalent bonds are formedas the result of a reaction between the azido group of the recurringunit of Formula (V) and the first surface of the substrate. Therefore,as described herein, a substrate having a first surface comprising apolymer with a recurring unit of Formula (V) covalently bonded thereto,should be understood to also include the polymer with a modifiedrecurring unit of the structure

showing the covalent bonding position with the substrate surface.

In some other embodiments, when the polymer is covalently attached tothe first surface of the substrate, at least one covalent bond is formedbetween the amino group of the recurring unit of Formula (V) and thefirst surface of the substrate. Therefore, as described herein, asubstrate having a first surface comprising a polymer with a recurringunit of Formula (V) covalently bonded thereto, should be understood toalso include the polymer with a modified recurring unit of the structure

showing the covalent bonding position with the substrate surface.

Some embodiments described herein are related to a grafted polymercomprising functionalized oligonucleotides covalently bonded to apolymer with a recurring unit of Formula (V) as described herein. Insome embodiments, the polymer may further comprise a recurring unit ofFormula (VIa) or (VIb), or both. In some embodiments, the polymer mayfurther comprise one or more recurring units of various differentpolymer backbones as described above.

In some embodiments, when functionalized oligonucleotides are covalentlybonded to the polymer, at least one covalent bond is formed as theresult of a reaction between the epoxy group of the recurring unit ofFormula (VIa) and a functionalized oligonucleotide. Therefore, asdescribed herein, a grafted polymer comprising functionalizedoligonucleotides covalently bonded to a polymer of a recurring unit ofFormula (V) and a recurring unit of Formula (VIa), should be understoodto also include the polymer with a modified recurring unit of thestructure

showing the covalent bonding position with the oligonucleotide.

Some embodiments described herein are related processes for immobilizinga grafted polymer to a first surface of a substrate, comprising:

-   -   providing a substrate having a first surface comprising a first        plurality of functional groups covalently attached thereto;    -   providing a grafted polymer comprising functionalized        oligonucleotides covalently bonded to a polymer, wherein the        polymer comprises a second plurality of functional groups; and    -   reacting the first plurality functional groups of the first        surface with the second plurality of functional groups of the        polymer such that the polymer is covalently bonded to the first        surface of the substrate.

In some embodiments of the methods described herein, when the surface istreated with functionalized silane comprising unsaturated moietiesselected from cycloalkenes, cycloalkynes, heterocycloalkenes,heterocycloalkynes; and the functionalized oligonucleotides comprisebicyclo[6.1.0] non-4-yne; then the polymer is not apoly(N-(5-azidoacetamidylpentyl) acrylamide-co-acrylamide) (PAZAM) ofthe following structure:

wherein n is an integer in the range of 1-20,000, and m is an integer inthe range of 1-100,000. In some such embodiments, the unsaturatedmoieties of the functionalized silane comprise norbornene.

Some embodiments described herein are related processes or methods forimmobilizing a polymer described herein to a first surface of asubstrate, comprising: providing a substrate having a first surfacecomprising a first plurality of functional groups covalently attachedthereto; providing a polymer described herein; and reacting the firstplurality functional groups of the first surface with the polymer suchthat the polymer is covalently bonded to the first surface of thesubstrate.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A to 1D show the Typhoon florescence image of the polymers coatedflow cell with norbornene silane monolayer surface and the related barchart of median Typhoon intensity of the polymers of Example 1 (Table2).

FIGS. 2A to 2D show the Typhoon florescence image of the polymers coatedflow cell with norbornene silane monolayer surface and the related barchart of median Typhoon intensity of the polymers of Example 1 (Table3).

FIG. 3A is a line and bar chart that illustrates the TET QC intensitydata (Table 4) after coating a norbornene surface with differentpolymers as listed in Example 1 (Table 2) and surface loss percentage asmeasured after a thermal Stress Test.

FIG. 3B is a line and bar chart that illustrates the TET QC intensitydata (Table 5) after coating an epoxy surface with different polymers aslisted in Example 1 (Table 3) and surface loss percentage as measuredafter a thermal Stress Test.

FIGS. 4A to 4D show the Typhoon florescence image of the polymers coatedflow cell with norbornene silane monolayer surface and the related barchart of median Typhoon intensity of the polymers of Example 1 (Table6).

FIG. 4E is a line and bar chart that illustrates the TET QC intensitydata (Table 7) after coating a norbornene surface with differentpolymers as listed Example 1 (Table 6) and surface loss percentage asmeasured after a thermal Stress Test.

FIG. 5 shows a series of NMR images of a reaction between norbornene anda bipyridyl tetrazine at different time points.

FIG. 6 shows a line graph of aUV-Vis absorption pattern of a reactionbetween bipyridyl tetrazine and bicyclo[6.1.0]non-4-yn-9-yl methanol.

FIG. 7 shows the coupling reaction between a grafted dendrimer and afunctionalized dendrimer with surface attachment groups.

DETAILED DESCRIPTION

The present application relates to nucleic acid-copolymers (for example,DNA-copolymers and processes for grafting such nucleic acid-copolymersto the surface of a substrate. Some embodiments of polymers used forpre-conjugation with single stranded DNA (“ssDNA”) primers includeacrylamide/azido-acrylamide/aminoethyl-acrylamide ternary copolymers,tetrazine modified polyacrylamide, and the reaction products ofpoly(glycidyl methacrylate) with amino-PEG-azide or amino-PEG-Boc-amide.The nucleic acid-copolymer can then be covalently attached to a surfaceof a substrate, in some instances, a silane functionalized surface of asubstrate, such as a surface of a flow cell or a surface of a moleculararray. The present disclosure also relates to methods of preparing suchnucleic acid-copolymer coated surfaces and methods of using substratescomprising such nucleic acid-copolymer coated surfaces insequencing-by-synthesis reactions.

Some embodiments relate to flow cells for performingsequencing-by-synthesis reactions that include functionalizedoligonucleotides pre-conjugated to a polymer described herein throughone or more functional moieties, such as bicyclo[6.1.0]non-4-yne,alkyne, amido or azido derivatized linkage. In some embodiments, theprimers are a P5 or P7 primer. The P5 and P7 primers are used on thesurface of commercial flow cells sold by Illumina Inc. for sequencing onthe HiSeq®, MiSeq®, NextSeq® and Genome Analyzer® platforms.

Further process and cost savings may be achieved by incorporatingquality control (QC) markers within the polymer along with the primerattachment. Analytical tests may be used to determine the quality andconsistency of the DNA-copolymer with or without QC markers, and usedagain to determine effectiveness of deposition when working with an openwafer format. These additional quality control checkpoints should reduceflow cell batch to batch variation to yield more consistent products,and also help narrow down where process deviation has occurred whenfailures during manufacturing appear.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art. The use of the term “including” as well as other forms, suchas “include”, “includes,” and “included,” is not limiting. The use ofthe term “having” as well as other forms, such as “have”, “has,” and“had,” is not limiting. As used in this specification, whether in atransitional phrase or in the body of the claim, the terms “comprise(s)”and “comprising” are to be interpreted as having an open-ended meaning.That is, the above terms are to be interpreted synonymously with thephrases “having at least” or “including at least.” For example, whenused in the context of a process, the term “comprising” means that theprocess includes at least the recited steps, but may include additionalsteps. When used in the context of a compound, composition, or device,the term “comprising” means that the compound, composition, or deviceincludes at least the recited features or components, but may alsoinclude additional features or components.

As used herein, common organic abbreviations are defined as follows:

-   -   Ac Acetyl    -   Ac₂O Acetic anhydride    -   APTS aminopropyl silane    -   APTES (3-aminopropyl)triethoxysilane    -   APTMS (3-aminopropyl)trimethoxysilane    -   aq. Aqueous    -   ATRP Atom-transfer radical polymerization    -   Azapa N-(5-azidoacetamidylpentyl) acrylamide    -   BCN Bicyclo[6.1.0] non-4-yne    -   Bn Benzyl    -   Brapa or BRAPA N-(5-bromoacetamidylpentyl) acrylamide    -   Bz Benzoyl    -   BOC or Boc tert-Butoxycarbonyl    -   Bu n-Butyl    -   cat. Catalytic    -   CMP Chemical mechanical polishing    -   CRP Controlled radical polymerization    -   CVD Chemical vapor deposition    -   ° C. Temperature in degrees Centigrade    -   dATP Deoxyadenosine triphosphate    -   dCTP Deoxycytidine triphosphate    -   dGTP Deoxyguanosine triphosphate    -   dTTP Deoxythymidine triphosphate    -   DCA Dichloroacetic acid    -   DCE 1,2-Dichloroethane    -   DCM Methylene chloride    -   DIEA Diisopropylethylamine    -   DIPEA Diisopropylethylamine    -   DMA Dimethylacetamide    -   DME Dimethoxyethane    -   DMF N,N′-Dimethylformamide    -   DMSO Dimethylsulfoxide    -   DPPA Diphenylphosphoryl azide    -   Et Ethyl    -   EtOAc or EA Ethyl acetate    -   g Gramme(s)    -   h or hr Hour(s)    -   iPr Isopropyl    -   KPi 10 mM potassium phosphate buffer at pH 7.0    -   KPS Potassium persulfate    -   IPA Isopropyl Alcohol    -   IPHA.HCl N-Isopropylhydroxylamine hydrochloride    -   m or min Minute(s)    -   MeOH Methanol    -   MeCN Acetonitrile    -   mL Milliliter(s)    -   NaN₃ Sodium Azide    -   NHS N-hydroxysuccinimide    -   NMP Nitroxide-mediated radical polymerisation    -   PAZAM poly(N-(5-azidoacetamidylpentyl) acrylamide-co-acrylamide)        of any acrylamide to Azapa ratio    -   PEG Polyethylene glycol    -   PG Protecting group    -   PGMA Poly(glycidyl methacrylate)    -   Ph Phenyl    -   ppt Precipitate    -   RAFT Reversible addition-fragmentation chain transfer        polymerisation    -   rt Room temperature    -   SFA Silane Free Acrylamide as defined in U.S. Pat. Pub. No.        2011/0059865    -   Sulfo-HSAB or SHSAB N-Hydroxysulfosuccinimidyl-4-azidobenzoate    -   TEA Triethylamine    -   Tert, t tertiary    -   THF Tetrahydrofuran    -   TEMED Tetramethylethylenediamine    -   YES Yield Engineering Systems    -   μL Microliter(s)

As used herein, the term “array” refers to a population of differentprobe molecules that are attached to one or more substrates such thatthe different probe molecules can be differentiated from each otheraccording to relative location. An array can include different probemolecules that are each located at a different addressable location on asubstrate. Alternatively or additionally, an array can include separatesubstrates each bearing a different probe molecule, wherein thedifferent probe molecules can be identified according to the locationsof the substrates on a surface to which the substrates are attached oraccording to the locations of the substrates in a liquid. Exemplaryarrays in which separate substrates are located on a surface include,without limitation, those including beads in wells as described, forexample, in U.S. Pat. No. 6,355,431 B1, US 2002/0102578 and PCTPublication No. WO 00/63437. Exemplary formats that can be used in theinvention to distinguish beads in a liquid array, for example, using amicrofluidic device, such as a fluorescent activated cell sorter (FACS),are described, for example, in U.S. Pat. No. 6,524,793. Further examplesof arrays that can be used in the invention include, without limitation,those described in U.S. Pat. Nos. 5,429,807; 5,436,327; 5,561,071;5,583,211; 5,658,734; 5,837,858; 5,874,219; 5,919,523; 6,136,269;6,287,768; 6,287,776; 6,288,220; 6,297,006; 6,291,193; 6,346,413;6,416,949; 6,482,591; 6,514,751 and 6,610,482; and WO 93/17126; WO95/11995; WO 95/35505; EP 742 287; and EP 799 897.

As used herein, the term “covalently attached” or “covalently bonded”refers to the forming of a chemical bonding that is characterized by thesharing of pairs of electrons between atoms. For example, a covalentlyattached polymer coating refers to a polymer coating that forms chemicalbonds with a functionalized surface of a substrate, as compared toattachment to the surface via other means, for example, adhesion orelectrostatic interaction. It will be appreciated that polymers that areattached covalently to a surface can also be bonded via means inaddition to covalent attachment.

As used herein, “C_(a) to C_(b)” or “C_(a-b)” in which “a” and “b” areintegers refer to the number of carbon atoms in the specified group.That is, the group can contain from “a” to “b”, inclusive, carbon atoms.Thus, for example, a “C₁ to C₄ alkyl” or “C₁₋₄ alkyl” group refers toall alkyl groups having from 1 to 4 carbons, that is, CH₃—, CH₃CH₂—,CH₃CH₂CH₂—, (CH₃)₂CH—, CH₃CH₂CH₂CH₂—, CH₃CH₂CH(CH₃)— and (CH₃)₃C—.

The term “halogen” or “halo,” as used herein, means any one of theradio-stable atoms of column 7 of the Periodic Table of the Elements,e.g., fluorine, chlorine, bromine, or iodine, with fluorine and chlorinebeing preferred.

As used herein, “alkyl” refers to a straight or branched hydrocarbonchain that is fully saturated (i.e., contains no double or triplebonds). The alkyl group may have 1 to 20 carbon atoms (whenever itappears herein, a numerical range such as “1 to 20” refers to eachinteger in the given range; e.g., “1 to 20 carbon atoms” means that thealkyl group may consist of 1 carbon atom, 2 carbon atoms, 3 carbonatoms, etc., up to and including 20 carbon atoms, although the presentdefinition also covers the occurrence of the term “alkyl” where nonumerical range is designated). The alkyl group may also be a mediumsize alkyl having 1 to 9 carbon atoms. The alkyl group could also be alower alkyl having 1 to 4 carbon atoms. The alkyl group may bedesignated as “C₁₋₄ alkyl” or similar designations. By way of exampleonly, “C₁₋₄ alkyl” indicates that there are one to four carbon atoms inthe alkyl chain, i.e., the alkyl chain is selected from the groupconsisting of methyl, ethyl, propyl, iso-propyl, n-butyl, iso-butyl,sec-butyl, and t-butyl. Typical alkyl groups include, but are in no waylimited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiarybutyl, pentyl, hexyl, and the like. The alkyl group can be optionallysubstituted.

As used herein, “alkoxy” refers to the formula —OR wherein R is an alkylas is defined above, such as “C₁₋₉ alkoxy”, including but not limited tomethoxy, ethoxy, n-propoxy, 1-methylethoxy (isopropoxy), n-butoxy,iso-butoxy, sec-butoxy, and tert-butoxy, and the like. The alkoxy groupcan be optionally substituted.

As used herein, “alkenyl” refers to a straight or branched hydrocarbonchain containing one or more double bonds. The alkenyl group may have 2to 20 carbon atoms, although the present definition also covers theoccurrence of the term “alkenyl” where no numerical range is designated.The alkenyl group may also be a medium size alkenyl having 2 to 9 carbonatoms. The alkenyl group could also be a lower alkenyl having 2 to 4carbon atoms. The alkenyl group may be designated as “C₂₋₄ alkenyl” orsimilar designations. By way of example only, “C₂₋₄ alkenyl” indicatesthat there are two to four carbon atoms in the alkenyl chain, i.e., thealkenyl chain is selected from the group consisting of ethenyl,propen-1-yl, propen-2-yl, propen-3-yl, buten-1-yl, buten-2-yl,buten-3-yl, buten-4-yl, 1-methyl-propen-1-yl, 2-methyl-propen-1-yl,1-ethyl-ethen-1-yl, 2-methyl-propen-3-yl, buta-1,3-dienyl,buta-1,2,-dienyl, and buta-1,2-dien-4-yl. Typical alkenyl groupsinclude, but are in no way limited to, ethenyl, propenyl, butenyl,pentenyl, and hexenyl, and the like. The alkenyl group can be optionallysubstituted.

As used herein, “alkynyl” refers to a straight or branched hydrocarbonchain containing one or more triple bonds. The alkynyl group may have 2to 20 carbon atoms, although the present definition also covers theoccurrence of the term “alkynyl” where no numerical range is designated.The alkynyl group may also be a medium size alkynyl having 2 to 9 carbonatoms. The alkynyl group could also be a lower alkynyl having 2 to 4carbon atoms. The alkynyl group may be designated as “C₂₋₄ alkynyl” orsimilar designations. By way of example only, “C₂₋₄ alkynyl” indicatesthat there are two to four carbon atoms in the alkynyl chain, i.e., thealkynyl chain is selected from the group consisting of ethynyl,propyn-1-yl, propyn-2-yl, butyn-1-yl, butyn-3-yl, butyn-4-yl, and2-butynyl. Typical alkynyl groups include, but are in no way limited to,ethynyl, propynyl, butynyl, pentynyl, and hexynyl, and the like. Thealkynyl group can be optionally substituted.

As used herein, “heteroalkyl” refers to a straight or branchedhydrocarbon chain containing one or more heteroatoms, that is, anelement other than carbon, including but not limited to, nitrogen,oxygen and sulfur, in the chain backbone. The heteroalkyl group may have1 to 20 carbon atoms, although the present definition also covers theoccurrence of the term “heteroalkyl” where no numerical range isdesignated. The heteroalkyl group may also be a medium size heteroalkylhaving 1 to 9 carbon atoms. The heteroalkyl group could also be a lowerheteroalkyl having 1 to 4 carbon atoms. The heteroalkyl group may bedesignated as “C₁₋₄ heteroalkyl” or similar designations. Theheteroalkyl group may contain one or more heteroatoms. By way of exampleonly, “C₁₋₄ heteroalkyl” indicates that there are one to four carbonatoms in the heteroalkyl chain and additionally one or more heteroatomsin the backbone of the chain.

As used herein, “alkylene” means a branched, or straight chain fullysaturated di-radical chemical group containing only carbon and hydrogenthat is attached to the rest of the molecule via two points ofattachment (i.e., an alkanediyl). The alkylene group may have 1 to20,000 carbon atoms, although the present definition also covers theoccurrence of the term alkylene where no numerical range is designated.The alkylene group may also be a medium size alkylene having 1 to 9carbon atoms. The alkylene group could also be a lower alkylene having 1to 4 carbon atoms. The alkylene group may be designated as “C₁₋₄alkylene” or similar designations. By way of example only, “C₁₋₄alkylene” indicates that there are one to four carbon atoms in thealkylene chain, i.e., the alkylene chain is selected from the groupconsisting of methylene, ethylene, ethan-1,1-diyl, propylene,propan-1,1-diyl, propan-2,2-diyl, 1-methyl-ethylene, butylene,butan-1,1-diyl, butan-2,2-diyl, 2-methyl-propan-1,1-diyl,1-methyl-propylene, 2-methyl-propylene, 1,1-dimethyl-ethylene,1,2-dimethyl-ethylene, and 1-ethyl-ethylene.

As used herein, the term “heteroalkylene” refers to an alkylene chain inwhich one or more skeletal atoms of the alkylene are selected from anatom other than carbon, e.g., oxygen, nitrogen, sulfur, phosphorus orcombinations thereof. The heteroalkylene chain can have a length of 2 to20,000. Exemplary heteroalkylenes include, but are not limited to,—OCH₂—, —OCH(CH₃)—, —OC(CH₃)₂—, —OCH₂CH₂—, —CH(CH₃)O—, —CH₂OCH₂—,—CH₂OCH₂CH₂—, —SCH₂—, -SCH(CH₃)—, —SC(CH₃)₂—, —SCH₂CH₂—, —CH₂SCH₂CH₂—,—NHCH₂—, —NHCH(CH₃)—, —NHC(CH₃)₂—, —NHCH₂CH₂—, —CH₂NHCH₂—,—CH₂NHCH₂CH₂—, and the like.

As used herein, “alkenylene” means a straight or branched chaindi-radical chemical group containing only carbon and hydrogen andcontaining at least one carbon-carbon double bond that is attached tothe rest of the molecule via two points of attachment. The alkenylenegroup may have 2 to 20,000 carbon atoms, although the present definitionalso covers the occurrence of the term alkenylene where no numericalrange is designated. The alkenylene group may also be a medium sizealkenylene having 2 to 9 carbon atoms. The alkenylene group could alsobe a lower alkenylene having 2 to 4 carbon atoms. The alkenylene groupmay be designated as “C₂₋₄ alkenylene” or similar designations. By wayof example only, “C₂₋₄ alkenylene” indicates that there are two to fourcarbon atoms in the alkenylene chain, i.e., the alkenylene chain isselected from the group consisting of ethenylene, ethen-1,1-diyl,propenylene, propen-1,1-diyl, prop-2-en-1,1-diyl, 1-methyl-ethenylene,but-1-enylene, but-2-enylene, but-1,3-dienylene, buten-1,1-diyl,but-1,3-dien-1,1-diyl, but-2-en-1,1-diyl, but-3-en-1,1-diyl,1-methyl-prop-2-en-1,1-diyl, 2-methyl-prop-2-en-1,1-diyl,1-ethyl-ethenylene, 1,2-dimethyl-ethenylene, 1-methyl-propenylene,2-methyl-propenylene, 3-methyl-propenylene, 2-methyl-propen-1,1-diyl,and 2,2-dimethyl-ethen-1,1-diyl.

As used herein, “alkynylene” means a straight or branched chaindi-radical chemical group containing only carbon and hydrogen andcontaining at least one carbon-carbon triple bond that is attached tothe rest of the molecule via two points of attachment.

The term “aromatic” refers to a ring or ring system having a conjugatedpi electron system and includes both carbocyclic aromatic (e.g., phenyl)and heterocyclic aromatic groups (e.g., pyridine). The term includesmonocyclic or fused-ring polycyclic (i.e., rings which share adjacentpairs of atoms) groups provided that the entire ring system is aromatic.

As used herein, “aryl” refers to an aromatic ring or ring system (i.e.,two or more fused rings that share two adjacent carbon atoms) containingonly carbon in the ring backbone. When the aryl is a ring system, everyring in the system is aromatic. The aryl group may have 6 to 18 carbonatoms, although the present definition also covers the occurrence of theterm “aryl” where no numerical range is designated. In some embodiments,the aryl group has 6 to 10 carbon atoms. The aryl group may bedesignated as “C₆₋₁₀ aryl,” “C₆ or C₁₀ aryl,” or similar designations.Examples of aryl groups include, but are not limited to, phenyl,naphthyl, azulenyl, and anthracenyl. The aryl group can be optionallysubstituted.

As used herein, “arylene” refers to an aromatic ring or ring systemcontaining only carbon and hydrogen that is attached to the rest of themolecule via two points of attachment.

An “aralkyl” or “arylalkyl” is an aryl group connected, as asubstituent, via an alkylene group, such as “C₇₋₁₄ aralkyl” and thelike, including but not limited to benzyl, 2-phenylethyl,3-phenylpropyl, and naphthylalkyl. In some cases, the alkylene group isa lower alkylene group (i.e., a C₁₋₄ alkylene group).

As used herein, “heteroaryl” refers to an aromatic ring or ring system(i.e., two or more fused rings that share two adjacent atoms) thatcontain(s) one or more heteroatoms, that is, an element other thancarbon, including but not limited to, nitrogen, oxygen and sulfur, inthe ring backbone. When the heteroaryl is a ring system, every ring inthe system is aromatic. The heteroaryl group may have 5-18 ring members(i.e., the number of atoms making up the ring backbone, including carbonatoms and heteroatoms), although the present definition also covers theoccurrence of the term “heteroaryl” where no numerical range isdesignated. In some embodiments, the heteroaryl group has 5 to 10 ringmembers or 5 to 7 ring members. The heteroaryl group may be designatedas “5-7 membered heteroaryl,” “5-10 membered heteroaryl,” or similardesignations. Examples of heteroaryl rings include, but are not limitedto, furyl, thienyl, phthalazinyl, pyrrolyl, oxazolyl, thiazolyl,imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, triazolyl,thiadiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl,quinolinyl, isoquinolinyl, benzimidazolyl, benzoxazolyl, benzothiazolyl,indolyl, isoindolyl, and benzothienyl. The heteroaryl group can beoptionally substituted.

As used herein, “heteroarylene” refers to an aromatic ring or ringsystem containing one or more heteroatoms in the ring backbone that isattached to the rest of the molecule via two points of attachment.

A “heteroaralkyl” or “heteroarylalkyl” is heteroaryl group connected, asa substituent, via an alkylene group. Examples include but are notlimited to 2-thienylmethyl, 3-thienylmethyl, furylmethyl, thienylethyl,pyrrolylalkyl, pyridylalkyl, isoxazolylalkyl, and imidazolylalkyl. Insome cases, the alkylene group is a lower alkylene group (i.e., a C₁₋₄alkylene group).

As used herein, “carbocyclyl” means a non-aromatic cyclic ring or ringsystem containing only carbon atoms in the ring system backbone. Whenthe carbocyclyl is a ring system, two or more rings may be joinedtogether in a fused, bridged or spiro-connected fashion. Carbocyclylsmay have any degree of saturation provided that at least one ring in aring system is not aromatic. Thus, carbocyclyls include cycloalkyls,cycloalkenyls, and cycloalkynyls. The carbocyclyl group may have 3 to 20carbon atoms, although the present definition also covers the occurrenceof the term “carbocyclyl” where no numerical range is designated. Thecarbocyclyl group may also be a medium size carbocyclyl having 3, 4, 5,6, 7, 8, 9 or 10 carbon atoms. The carbocyclyl group could also be acarbocyclyl having 3 to 6 carbon atoms. The carbocyclyl group may bedesignated as “C₃₋₆ carbocyclyl” or similar designations. Examples ofcarbocyclyl rings include, but are not limited to, cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, 2,3-dihydro-indene,bicycle[2.2.2]octanyl, adamantyl, and spiro[4.4]nonanyl.

As used herein, “cycloalkyl” means a fully saturated carbocyclyl ring orring system. Examples include cyclopropyl, cyclobutyl, cyclopentyl, andcyclohexyl.

As used herein, “cycloalkylene” means a fully saturated carbocyclyl ringor ring system that is attached to the rest of the molecule via twopoints of attachment.

As used herein, “cycloalkenyl” or “cycloalkene” means a carbocyclyl ringor ring system having at least one double bond, wherein no ring in thering system is aromatic. An example is cyclohexenyl or cyclohexene.Another example is norbornene or norbornenyl.

As used herein, “heterocycloalkenyl” or “heterocycloalkene” means acarbocyclyl ring or ring system with at least one heteroatom in ringbackbone, having at least one double bond, wherein no ring in the ringsystem is aromatic. In some embodiments, heterocycloalkenyl orheterocycloalkene ring or ring system is 3-membered, 4-membered,5-membered, 6-membered, 7-membered, 8-membered, 9 membered, or 10membered.

As used herein, “cycloalkynyl” or “cycloalkyne” means a carbocyclyl ringor ring system having at least one triple bond, wherein no ring in thering system is aromatic. An example is cyclooctyne. Another example isbicyclononyne.

As used herein, “heterocycloalkynyl” or “heterocycloalkyne” means acarbocyclyl ring or ring system with at least one heteroatom in ringbackbone, having at least one triple bond, wherein no ring in the ringsystem is aromatic. In some embodiments, heterocycloalkynyl orheterocycloalkyne ring or ring system is 3-membered, 4-membered,5-membered, 6-membered, 7-membered, 8-membered, 9 membered, or 10membered.

As used herein, “heterocyclyl” means a non-aromatic cyclic ring or ringsystem containing at least one heteroatom in the ring backbone.Heterocyclyls may be joined together in a fused, bridged orspiro-connected fashion. Heterocyclyls may have any degree of saturationprovided that at least one ring in the ring system is not aromatic. Theheteroatom(s) may be present in either a non-aromatic or aromatic ringin the ring system. The heterocyclyl group may have 3 to 20 ring members(i.e., the number of atoms making up the ring backbone, including carbonatoms and heteroatoms), although the present definition also covers theoccurrence of the term “heterocyclyl” where no numerical range isdesignated. The heterocyclyl group may also be a medium sizeheterocyclyl having 3 to 10 ring members. The heterocyclyl group couldalso be a heterocyclyl having 3 to 6 ring members. The heterocyclylgroup may be designated as “3-6 membered heterocyclyl” or similardesignations. In preferred six membered monocyclic heterocyclyls, theheteroatom(s) are selected from one up to three of O, N or S, and inpreferred five membered monocyclic heterocyclyls, the heteroatom(s) areselected from one or two heteroatoms selected from O, N, or S. Examplesof heterocyclyl rings include, but are not limited to, azepinyl,acridinyl, carbazolyl, cinnolinyl, dioxolanyl, imidazolinyl,imidazolidinyl, morpholinyl, oxiranyl, oxepanyl, thiepanyl, piperidinyl,piperazinyl, dioxopiperazinyl, pyrrolidinyl, pyrrolidonyl,pyrrolidionyl, 4-piperidonyl, pyrazolinyl, pyrazolidinyl, 1,3-dioxinyl,1,3-dioxanyl, 1,4-dioxinyl, 1,4-dioxanyl, 1,3-oxathianyl,1,4-oxathiinyl, 1,4-oxathianyl, 2H-1,2-oxazinyl, trioxanyl,hexahydro-1,3,5-triazinyl, 1,3-dioxolyl, 1,3-dioxolanyl, 1,3-dithiolyl,1,3-dithiolanyl, isoxazolinyl, isoxazolidinyl, oxazolinyl, oxazolidinyl,oxazolidinonyl, thiazolinyl, thiazolidinyl, 1,3-oxathiolanyl, indolinyl,isoindolinyl, tetrahydrofuranyl, tetrahydropyranyl,tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydro-1,4-thiazinyl,thiamorpholinyl, dihydrobenzofuranyl, benzimidazolidinyl, andtetrahydroquinoline.

As used herein, “heterocyclylene” means a non-aromatic cyclic ring orring system containing at least one heteroatom that is attached to therest of the molecule via two points of attachment.

As used herein, “acyl” refers to —C(═O)R, wherein R is hydrogen, C₁₋₆alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₇ carbocyclyl, C₆₋₁₀ aryl, 5-10membered heteroaryl, and 5-10 membered heterocyclyl, as defined herein.Non-limiting examples include formyl, acetyl, propanoyl, benzoyl, andacryl.

An “O-carboxy” group refers to a “—OC(═O)R” group in which R is selectedfrom hydrogen, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₇ carbocyclyl,C₆₋₁₀ aryl, 5-10 membered heteroaryl, and 5-10 membered heterocyclyl, asdefined herein.

A “C-carboxy” group refers to a “—C(═O)OR” group in which R is selectedfrom hydrogen, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₇ carbocyclyl,C₆₋₁₀ aryl, 5-10 membered heteroaryl, and 5-10 membered heterocyclyl, asdefined herein. A non-limiting example includes carboxyl (i.e.,—C(═O)OH).

An “acetal” group refers to RC(H)(OR′)₂, in which R and R′ areindependently selected from hydrogen, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆alkynyl, C₃₋₇ carbocyclyl, C₆₋₁₀ aryl, 5-10 membered heteroaryl, and5-10 membered heterocyclyl, as defined herein.

A “cyano” group refers to a “—CN” group.

A “sulfinyl” group refers to an “—S(=O)R” group in which R is selectedfrom hydrogen, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₇ carbocyclyl,C₆₋₁₀ aryl, 5-10 membered heteroaryl, and 5-10 membered heterocyclyl, asdefined herein.

A “sulfonyl” group refers to an “—SO₂R” group in which R is selectedfrom hydrogen, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₇ carbocyclyl,C₆₋₁₀ aryl, 5-10 membered heteroaryl, and 5-10 membered heterocyclyl, asdefined herein.

An “S-sulfonamido” group refers to a “—SO₂NR_(A)R_(B)” group in whichR_(A) and R_(B) are each independently selected from hydrogen, C₁₋₆alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₇ carbocyclyl, C₆₋₁₀ aryl, 5-10membered heteroaryl, and 5-10 membered heterocyclyl, as defined herein.

An “N-sulfonamido” group refers to a “—N(R_(A))SO₂R_(B)” group in whichR_(A) and R_(B) are each independently selected from hydrogen, C₁₋₆alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₇ carbocyclyl, C₆₋₁₀ aryl, 5-10membered heteroaryl, and 5-10 membered heterocyclyl, as defined herein.

An “O-carbamyl” group refers to a “—OC(═O)NR_(A)R_(B)” group in whichR_(A) and R_(B) are each independently selected from hydrogen, C₁₋₆alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₇ carbocyclyl, C₆₋₁₀ aryl, 5-10membered heteroaryl, and 5-10 membered heterocyclyl, as defined herein.

An “N-carbamyl” group refers to an “—N(R_(A))OC(═O)R_(B)” group in whichR_(A) and R_(B) are each independently selected from hydrogen, C₁₋₆alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₇ carbocyclyl, C₆₋₁₀ aryl, 5-10membered heteroaryl, and 5-10 membered heterocyclyl, as defined herein.

A “C-amido” group refers to a “—C(═O)NR_(A)R_(B)” group in which R_(A)and R_(B) are each independently selected from hydrogen, C₁₋₆ alkyl,C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₇ carbocyclyl, C₆₋₁₀ aryl, 5-10 memberedheteroaryl, and 5-10 membered heterocyclyl, as defined herein.

An “N-amido” group refers to a “—N(R_(A))C(═O)R_(B)” group in whichR_(A) and R_(B) are each independently selected from hydrogen, C₁₋₆alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₇ carbocyclyl, C₆₋₁₀ aryl, 5-10membered heteroaryl, and 5-10 membered heterocyclyl, as defined herein.

An “amino” group refers to a “—NR_(A)R_(B)” group in which R_(A) andR_(B) are each independently selected from hydrogen, C₁₋₆ alkyl, C₂₋₆alkenyl, C₂₋₆ alkynyl, C₃₋₇ carbocyclyl, C₆₋₁₀ aryl, 5-10 memberedheteroaryl, and 5-10 membered heterocyclyl, as defined herein. Anon-limiting example includes free amino (i.e., —NH₂).

The term “hydrazine” or “hydrazinyl” as used herein refers to a —NHNH₂group.

The term “hydrazone” or “hydrazonyl” as used herein refers to a

group.

The term “formyl” as used herein refers to a —C(O)H group.

The term “hydroxy” as used herein refers to a —OH group.

The term “azido” as used herein refers to a —N₃ group.

The term “thiol” as used herein refers to a —SH group.

The term “glycidyl ether” as used herein refers to

The term “epoxy” as used herein refers to

The term “ester” as used herein refers to R—C(═O)O—R′, wherein R and R′can be independently alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl,(heteroalicyclyl)alkyl, or optionally substituted variants thereof.

The term “carboxylic acid” or “carboxyl” as used herein refers to—C(O)OH.

As used herein, the term “tetrazine” or “tetrazinyl” refers tosix-membered heteroaryl group comprising four nitrogen atoms. Tetrazinecan be optionally substituted.

As used herein, a substituted group is derived from the unsubstitutedparent group in which there has been an exchange of one or more hydrogenatoms for another atom or group. Unless otherwise indicated, when agroup is deemed to be “substituted,” it is meant that the group issubstituted with one or more substituents independently selected fromC₁-C₆ alkyl, C₁-C₆ alkenyl, C₁-C₆ alkynyl, C₁-C₆ heteroalkyl, C₃-C₇carbocyclyl (optionally substituted with halo, C₁-C₆ alkyl, C₁-C₆alkoxy, C₁-C₆ haloalkyl, and C₁-C₆ haloalkoxy),C₃-C₇-carbocyclyl-C₁-C₆-alkyl (optionally substituted with halo, C₁-C₆alkyl, C₁-C₆ alkoxy, C₁-C₆ haloalkyl, and C₁-C₆ haloalkoxy), 5-10membered heterocyclyl (optionally substituted with halo, C₁-C₆ alkyl,C₁-C₆ alkoxy, C₁-C₆ haloalkyl, and C₁-C₆ haloalkoxy), 5-10 memberedheterocyclyl-C₁-C₆-alkyl (optionally substituted with halo, C₁-C₆ alkyl,C₁-C₆ alkoxy, C₁-C₆ haloalkyl, and C₁-C₆ haloalkoxy), aryl (optionallysubstituted with halo, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ haloalkyl, andC₁-C₆ haloalkoxy), aryl(C₁-C₆)alkyl (optionally substituted with halo,C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ haloalkyl, and C₁-C₆ haloalkoxy), 5-10membered heteroaryl (optionally substituted with halo, C₁-C₆ alkyl,C₁-C₆ alkoxy, C₁-C₆ haloalkyl, and C₁-C₆ haloalkoxy), 5-10 memberedheteroaryl(C₁-C₆)alkyl (optionally substituted with halo, C₁-C₆ alkyl,C₁-C₆ alkoxy, C₁-C₆ haloalkyl, and C₁-C₆ haloalkoxy), halo, cyano,hydroxy, C₁-C₆ alkoxy, C₁-C₆ alkoxy(C₁-C₆)alkyl (i.e., ether), aryloxy,sulfhydryl (mercapto), halo(C₁-C₆)alkyl (e.g., —CF₃), halo(C₁-C₆)alkoxy(e.g., —OCF₃), C₁-C₆ alkylthio, arylthio, amino, amino(C₁-C₆)alkyl,nitro, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido,N-amido, S-sulfonamido, N-sulfonamido, C-carboxy, O-carboxy, acyl,cyanato, isocyanato, thiocyanato, isothiocyanato, sulfinyl, sulfonyl,and oxo (═O). Wherever a group is described as “optionally substituted”that group can be substituted with the above substituents.

It is to be understood that certain radical naming conventions caninclude either a mono-radical or a di-radical, depending on the context.For example, where a substituent requires two points of attachment tothe rest of the molecule, it is understood that the substituent is adi-radical. For example, a substituent identified as alkyl that requirestwo points of attachment includes di-radicals such as —CH₂—, —CH₂CH₂—,—CH₂CH(CH₃)CH₂—, and the like. Other radical naming conventions clearlyindicate that the radical is a di-radical such as “alkylene” or“alkenylene.”

Wherever a substituent is depicted as a di-radical (i.e., has two pointsof attachment to the rest of the molecule), it is to be understood thatthe substituent can be attached in any directional configuration unlessotherwise indicated. Thus, for example, a substituent depicted as -AE-or

includes the substituent being oriented such that the A is attached atthe leftmost attachment point of the molecule as well as the case inwhich A is attached at the rightmost attachment point of the molecule.

Where the compounds disclosed herein have at least one stereocenter,they may exist as individual enantiomers and diastereomers or asmixtures of such isomers, including racemates. Separation of theindividual isomers or selective synthesis of the individual isomers isaccomplished by application of various methods which are well known topractitioners in the art. Unless otherwise indicated, all such isomersand mixtures thereof are included in the scope of the compoundsdisclosed herein. Furthermore, compounds disclosed herein may exist inone or more crystalline or amorphous forms. Unless otherwise indicated,all such forms are included in the scope of the compounds disclosedherein including any polymorphic forms. In addition, some of thecompounds disclosed herein may form solvates with water (i.e., hydrates)or common organic solvents. Unless otherwise indicated, such solvatesare included in the scope of the compounds disclosed herein.

As used herein, a “nucleotide” includes a nitrogen containingheterocyclic base, a sugar, and one or more phosphate groups. They canbe monomeric units (whether precursors or linked monomers) of a nucleicacid sequence. In RNA, the sugar is a ribose, and in DNA a deoxyribose,i.e. a sugar lacking a hydroxyl group that is present at the 2′ positionin ribose. The nitrogen containing heterocyclic base can be purine orpyrimidine base. Purine bases include adenine (A) and guanine (G), andmodified derivatives or analogs thereof. Pyrimidine bases includecytosine (C), thymine (T), and uracil (U), and modified derivatives oranalogs thereof. The C-1 atom of deoxyribose is bonded to N-1 of apyrimidine or N-9 of a purine.

As used herein, a “nucleoside” is structurally similar to a nucleotide,but lacks any phosphate moieties at the 5′ position. The term“nucleoside” is used herein in its ordinary sense as understood by thoseskilled in the art. Examples include, but are not limited to, aribonucleoside comprising a ribose moiety and a deoxyribonucleo sidecomprising a deoxyribose moiety. A modified pentose moiety is a pentosemoiety in which an oxygen atom has been replaced with a carbon and/or acarbon has been replaced with a sulfur or an oxygen atom. A “nucleoside”is a monomer that can have a substituted base and/or sugar moiety.Additionally, a nucleoside can be incorporated into larger DNA and/orRNA polymers and oligomers.

As used herein, the term “polynucleotide” refers to nucleic acids ingeneral, including DNA (e.g. genomic DNA cDNA), RNA (e.g. mRNA),synthetic oligonucleotides and synthetic nucleic acid analogs.Polynucleotides may include natural or non-natural bases, orcombinations thereof and natural or non-natural backbone linkages, e.g.phosphorothioates, PNA or 2′-O-methyl-RNA, or combinations thereof.

As used herein, the term “primer” is defined as a nucleic acid having asingle strand with a free 3′ OH group. A primer can also have amodification at the 5′ terminus to allow a coupling reaction or tocouple the primer to another moiety. The primer length can be any numberof bases long and can include a variety of non-natural nucleotides. Asused herein, “BCN primer” or “BCN modified primer” refers to a primercomprising covalently attached bicyclo[6.1.0] non-4-yne at the 5′terminus.

As used herein, the term “silane” refers to an organic or inorganiccompound containing one or more silicon atoms. Non-limiting example ofan inorganic silane compound is SiH₄, or halogenated SiH₄ where hydrogenis replaced by one or more halogen atoms. Non-limiting example of anorganic silane compound is X—R^(C)—Si(OR^(D))₃, wherein X is anon-hydrolyzable organic group, such as amino, vinyl, epoxy,methacrylate, sulfur, alkyl, alkenyl, alkynyl; R^(C) is a spacer, forexample —(CH₂)_(n)—, wherein n is 0 to 1000; R^(D) is selected fromhydrogen, optionally substituted alkyl, optionally substituted alkenyl,optionally substituted alkynyl, optionally substituted carbocyclyl,optionally substituted aryl, optionally substituted 5-10 memberedheteroaryl, and optionally substituted 5-10 membered heterocyclyl, asdefined herein. As used herein, the term “silane” can comprise mixturesof different silane compounds.

As used herein, the term “polymer” refers to a molecule composed of manyrepeated subunits or recurring units. Non-limiting examples of polymerstructures include linear, branched, or hyper-branched polymers.Non-limiting examples of linear polymers comprising block copolymers orrandom copolymers. Non-limiting examples of branched polymers includestar polymers, star-shaped or star-block polymers comprising bothhydrophobic and hydrophilic segments, H-shaped polymers comprising bothhydrophobic and hydrophilic segments, dumbbell shaped polymers, combpolymers, brush polymers, dendronized polymers, ladders, and dendrimers.The polymers described herein can also be in the form of polymernanoparticles. Other examples of polymer architectures include, but notlimited to ring block polymers, coil-cycle-coil polymers, etc.

As used herein, the prefixes “photo” or “photo-” mean relating to lightor electromagnetic radiation. The term can encompass all or part of theelectromagnetic spectrum including, but not limited to, one or more ofthe ranges commonly known as the radio, microwave, infrared, visible,ultraviolet, X-ray or gamma ray parts of the spectrum. The part of thespectrum can be one that is blocked by a metal region of a surface suchas those metals set forth herein. Alternatively or additionally, thepart of the spectrum can be one that passes through an interstitialregion of a surface such as a region made of glass, plastic, silica, orother material set forth herein. In particular embodiments, radiationcan be used that is capable of passing through a metal. Alternatively oradditionally, radiation can be used that is masked by glass, plastic,silica, or other material set forth herein.

As used herein, the term “YES method” refers to the chemical vapordeposition tool provided by Yield Engineering Systems (“YES”) withchemical vapor deposition process developed by Illumina, Inc. Itincludes three different vapor deposition systems. The automatedYES-VertaCoat silane vapor system is designed for volume production witha flexible wafer handling module that can accommodate 200 or 300 mmwafers. The manual load YES-1224P Silane Vapor System is designed forversatile volume production with its configurable large capacitychambers. Yes-LabKote is a low-cost, tabletop version that is ideal forfeasibility studies and for R&D.

As used herein, the term “percent surface remaining” can refer to theintensity measured using a TET QC to stain the P5/P7 surface primers.The P5 and P7 primers are used on the surface of commercial flow cellssold by Illumina Inc. for sequencing on the HiSeq®, MiSeq®, GenomeAnalyzer® and NextSeq® platforms. The primer sequences are described inU.S. Pat. Pub. No. 2011/0059865 A1, which is incorporated herein byreference. The P5 and P7 primer sequences comprise the following:

Paired End Set:

P5: paired end 5′→3′ SEQ ID NO: 1 AATGATACGGCGACCACCGAGAUCTACACP7: paired end 5′→3′ SEQ ID NO: 2 CAAGCAGAAGACGGCATACGAG*AT

Single Read Set:

P5: single read: 5′→3′ SEQ ID NO: 3 AATGATACGGCGACCACCGAP7: single read 5′→3′ SEQ ID NO: 4 CAAGCAGAAGACGGCATACGA

In some embodiments, the P5 and P7 primers may comprise a linker orspacer at the 5′ end. Such linker or spacer may be included in order topermit cleavage, or to confer some other desirable property, for exampleto enable covalent attachment to a polymer or a solid support, or to actas spacers to position the site of cleavage an optimal distance from thesolid support. In certain cases, 10 spacer nucleotides may be positionedbetween the point of attachment of the P5 or P7 primers to a polymer ora solid support. In some embodiments polyT spacers are used, althoughother nucleotides and combinations thereof can also be used. In oneembodiment, the spacer is a 10T spacer. TET is a dye labeledoligonucleotide having complimentary sequence to the P5/P7 primers. TETcan be hybridized to the P5/P7 primers on a surface; the excess TET canbe washed away, and the attached dye concentration can be measured byfluorescence detection using a scanning instrument such as a TyphoonScanner (General Electric).

Polymers and DNA-Copolymers

Polymers and Nucleic Acid-Copolymers with Recurring Units of Formulae(I) and (II)

Some embodiments described herein are related to a polymer for surfacefunctionalization, comprising a recurring unit of Formula (I) and arecurring unit of Formula (II) as described above.

wherein each R^(1a), R^(2a), R^(1b) and R^(2b) is independently selectedfrom hydrogen, optionally substituted alkyl or optionally substitutedphenyl; each R^(3a) and R^(3b) is independently selected from hydrogen,optionally substituted alkyl, optionally substituted phenyl, oroptionally substituted C₇₋₁₄ aralkyl; and each L¹ and L² isindependently selected from an optionally substituted alkylene linker oran optionally substituted heteroalkylene linker.

In some embodiments, R^(1a) is hydrogen. In some embodiments, R^(2a) ishydrogen. In some embodiments, R^(3a) is hydrogen. In some embodiments,R^(1a) is selected from hydrogen or optionally substituted alkyl,preferably C₁₋₆ alkyl and each of R^(2a) and R^(3a) is hydrogen. In someembodiments, each of R¹, R^(2a) and R^(3a) is hydrogen. In someembodiments, R^(1b) is hydrogen. In some embodiments, R^(2b) ishydrogen. In some embodiments, R^(3b) is hydrogen. In some embodiments,R^(1b) is selected from hydrogen or optionally substituted alkyl,preferably C₁₋₆ alkyl and each of R^(2b) and R^(3b) is hydrogen. In someembodiments, each of R^(1b), R^(2b) and R^(3b) is hydrogen. In someembodiments, L¹ is an optionally substituted alkylene. In some suchembodiments, L¹ is optionally substituted methylene. In some otherembodiments, L¹ is optionally substituted ethylene. In some furtherembodiments, L¹ is optionally substituted propylene. In someembodiments, L¹ is an optionally substituted heteroalkylene linker. Insome such embodiments, L¹ is —(CH₂)m-NH—(CH₂)n- optionally substitutedwith one or more oxo groups, and wherein each m and n is an integerindependently selected from 1 to 10. In some embodiments, L² is anoptionally substituted alkylene. In some such embodiments, L² isoptionally substituted methylene. In some other embodiments, L² isoptionally substituted ethylene. In some further embodiments, L² isoptionally substituted propylene. In some embodiments, the recurringunit of Formula (I) is also represented by Formula (Ia) or (Ib) andFormula (II) is also represented by Formula (IIa):

wherein each R^(1a) and R^(1b) is selected from hydrogen or methyl. Insome embodiments, the polymer comprises recurring units of Formula (Ia)and (IIa). In some other embodiments, the polymer comprises recurringunits of Formula (Ib) and (IIa). In some embodiments of the recurringunit of Formula (II) or (IIa), the amino functional group is in the formof an inorganic salt, for example, hydrochloride salt. In someembodiments, the recurring units of Formulae (I) and (II) are about 1:1in molar ratio. In some such embodiments, the recurring units ofFormulae (Ia) and (IIa) are about 1:1 in molar ratio. In some suchembodiments, the recurring units of Formulae (Ib) and (IIa) are about1:1 in molar ratio.

In some embodiments, the polymer may further comprise one or morerecurring units selected from the group consisting of polyacrylamides,polyacrylates, polyurethanes, polysiloxanes, silicones, polyacroleins,polyphosphazenes, polyisocyanates, poly-ols, and polysaccharides, orcombinations thereof. In some such embodiments, the polymer may furthercomprising one or more recurring units of polyacrylamide of Formula(IIIa) or (IIIb) or both:

wherein each R^(4a), R^(4b) and R^(5b) is selected from hydrogen or C₁₋₃alkyl; and each R^(5a), R^(6a), R^(6b) and R^(7b) is independentlyselected from hydrogen, optionally substituted C₁₋₆ alkyl or optionallysubstituted phenyl. In some embodiments, each R^(4a), R^(4b) and R^(5b)is selected from hydrogen or methyl. In some embodiments, R^(6b) andR^(7b) are both hydrogen. In some embodiments, at least one of R^(5a) orR^(6a) is hydrogen. In some such embodiments, both R^(5a) and R^(6a) arehydrogen. In some other embodiments, at least one of R^(5a) or R^(6a) ismethyl. In some such embodiments, both R^(5a) and R^(6a) are methyl. Insome such embodiments, the recurring units of Formula (IIIa) is alsorepresented by (IIIa1), (IIIa2) or (IIIa3):

In some such embodiments, the recurring unit of Formula (IIIb) is alsorepresented by (IIIb1):

In some specific embodiments, the polymer comprises recurring units ofFormulae (Ib), (IIa) and (IIIa). In some further embodiments, thepolymer comprises recurring units of Formulae (Ib), (IIa), (IIIa) and(IIIb). In some such embodiments, the mole percent of Formula (IIIa) isfrom about 85% to about 90%. In some such embodiments, the mole percentof Formulae (Ib) and (IIa) is about 5% each. In one embodiment, thepolymer comprises recurring units of Formulae (IIIa1), (Ib) and (IIa) inthe mole percent of about 90% to about 5% to about 5%. In anotherembodiment, the polymer comprises recurring units of Formulae (IIIa1),(Ib) and (IIa) in the molar percent ratio of about 85% to about 5% toabout 10%. In yet another embodiment, the polymer comprises recurringunits of Formulae (IIIa2), (Ib) and (IIa) in the molar percent ratio ofabout 90% to about 5% to about 5%. In some further embodiments, thepolymer may further comprise about 0.5 mol % to about 2 mol % of arecurring unit of Formula (IIIb1).

Some embodiments described herein are related to a grafted polymercomprising functionalized oligonucleotides covalently bonded to apolymer with a recurring unit of Formula (I) and a recurring unit ofFormula (II) as described herein. In some embodiments, the polymer mayfurther comprise one or more recurring units of various differentpolymer backbones as described above, for example, one or more recurringunits of polyacrylamide of Formula (IIIa) or (IIIb) or both. In someembodiments, the covalent bonding between the functionalizedoligonucleotide and the polymer comprises the structure moiety

or combinations thereof, wherein * indicates the point of connectionwith the functionalized oligonucleotide. In some such embodiments, thecovalent bonding between the functionalized oligonucleotide and thepolymer comprises structure moiety

In some other such embodiments, the covalent bonding between thefunctionalized oligonucleotide and the polymer comprises structuremoiety

In some such embodiments, the covalent bonding between thefunctionalized oligonucleotide and the polymer comprises structuremoiety

In some embodiments, the grafted polymer is prepared by reacting one ormore functional moieties of the functionalized oligonucleotide with thepolymer, said one or more functional moieties comprise or are selectedfrom alkynes, cycloalkenes, cycloalkynes, heterocycloalkenes,heterocycloalkynes, or optionally substituted variants or combinationsthereof. In some such embodiments, said one or more functional moietiescomprise alkyne or are selected from alkyne. In some other embodiments,said one or more functional moieties comprise or are selected fromnorbornene, cyclooctyne, or bicyclononyne, or optionally substitutedvariants or combinations thereof. In one embodiment, the bicyclononyneis bicyclo[6.1.0]non-4-yne. In some such embodiments, the graftedpolymer is prepared by reacting the azido groups of the polymer withsaid one or more functional moieties of the functionalizedoligonucleotides, for example, bicyclo[6.1.0]non-4-yne.

Polymers and Nucleic Acid-Copolymers with Recurring Units of Formula(IV)

Some embodiments described herein are related to a polymer for surfacefunctionalization, comprising a recurring unit of Formula (IV) asdescribed above:

wherein each R^(1c) and R^(2c) is independently selected from hydrogen,optionally substituted alkyl or optionally substituted phenyl; R^(3c) isselected from hydrogen, optionally substituted alkyl, optionallysubstituted phenyl, or optionally substituted C₇₋₁₄ aralkyl; Ar isselected from an optionally substituted C₆₋₁₀ aryl or an optionallysubstituted 5 or 6 membered heteroaryl; R^(A) is optionally substitutedtetrazine; and L³ is selected from a single bond, an optionallysubstituted alkylene linker or an optionally substituted heteroalkylenelinker.

In some embodiments, R^(1c) is hydrogen. In some embodiments, R^(2c) ishydrogen. In some embodiments, R^(3c) is hydrogen. In some embodiments,R^(1c) is selected from hydrogen or optionally substituted alkyl,preferably C₁₋₆ alkyl and each R^(2c) and R^(3c) is hydrogen. In someembodiments, each R^(1c), R^(2c) and R^(3c) is hydrogen. In someembodiments, Ar is an optionally substituted phenyl. In someembodiments, L³ is a single bond. In some embodiments, the recurringunit of Formula (IV) is also represented by Formula (IVa):

wherein R^(1c) is selected from hydrogen or methyl.

In some embodiments, the polymer may further comprise one or morerecurring units selected from the group consisting of polyacrylamides,polyacrylates, polyurethanes, polysiloxanes, silicones, polyacroleins,polyphosphazenes, polyisocyanates, poly-ols, and polysaccharides, orcombinations thereof. In some such embodiments, the polymer may furthercomprising one or more recurring units of polyacrylamide of Formula(IIIa) or (IIIb) with the structure shown above.

Some embodiments described herein are related to a grafted polymercomprising functionalized oligonucleotides covalently bonded to apolymer with a recurring unit of Formula (IV) as described herein. Insome embodiments, the polymer may further comprise one or more recurringunits of various different polymer backbones as described above, forexample, one or more recurring units of polyacrylamide of Formula (IIIa)or (IIIb) or both. In some embodiments, the covalent bonding between thefunctionalized oligonucleotide and the polymer comprises the structuremoiety

or combinations thereof, and wherein * indicates the point of connectionof the polymer with the functionalized oligonucleotide. In some suchembodiments, the covalent bonding between the functionalizedoligonucleotide and the polymer comprises structure moiety

In some other such embodiments, the covalent bonding between thefunctionalized oligonucleotide and the polymer comprises structuremoiety

In some embodiments, the grafted polymer is prepared by reacting one ormore functional moieties of the functionalized oligonucleotide with thepolymer, said one or more functional moieties comprise, or are selectedfrom, alkynes, cycloalkenes, cycloalkynes, heterocycloalkenes,heterocycloalkynes, or optionally substituted variants or combinationsthereof. In some such embodiments, the one or more functional moietiesmay include or be selected from norbornene, cyclooctyne, orbicyclononyne, or optionally substituted variants or combinationsthereof. In one embodiment, the bicyclononyne isbicyclo[6.1.0]non-4-yne. In some such embodiments, the grafted polymeris prepared by reacting the tetrazine groups of the polymer with saidone or more functional moieties of the functionalized oligonucleotides,for example, bicyclo[6.1.0]non-4-yne.

Polymers and Nucleic Acid-Copolymers with Recurring Units of Formula (V)

Some embodiments described herein are related to a polymer for surfacefunctionalization, comprising a recurring unit of Formula (V) asdescribed above:

wherein each R^(1d) and R^(2d) is independently selected from hydrogen,optionally substituted alkyl or optionally substituted phenyl; eachR^(3d) is selected from hydrogen, optionally substituted alkyl,optionally substituted phenyl, or optionally substituted C₇₋₁₄ aralkyl;R^(B) is selected from azido, optionally substituted amino,Boc-protected amino, hydroxy, thiol, alkynyl, alkenyl, halo, epoxy,tetrazine or aldehyde; each L⁴ and L⁵ is independently selected from anoptionally substituted alkylene linker or an optionally substitutedheteroalkylene linker.

In some embodiments, R^(1d) is alkyl group, preferably C₁₋₆ alkyl. Insome such embodiments, R^(1d) is methyl. In some other embodiments,R^(1d) is hydrogen. In some embodiments, eR^(2d) is hydrogen. In someembodiments, R^(3d) is hydrogen. In some embodiments, each R^(2d) andR^(3d) is hydrogen. In some embodiments, R^(B) is selected from azido,amino or Boc-protected amino, or combinations thereof. In someembodiments, L⁴ is an optionally substituted alkylene linker. In somesuch embodiments, L⁴ is a methylene linker. In some embodiments, L⁵ isan optionally substituted heteroalkylene linker. In some suchembodiments, L⁵ is

and wherein n is an integer of 1 to 50. In some embodiments, therecurring unit of Formula (V) is also represented by Formula (Va) or(Vb):

wherein each R^(1d) is independently selected from hydrogen or methyl.In some such embodiments, n is an integer of 1 to 20. In some suchembodiments, n is an integer of 1 to 10. In some such embodiments, n isan integer of 1 to 5. In one embodiment, n is 3. In some embodiments,the polymer comprises both Formulae (Va) and (Vb).

In some embodiments, the polymer may further comprise a recurring unitof Formula (VIa) or (VIb), or both:

wherein each R^(1c), R^(2c), R^(1f) and R^(2f) is independently selectedfrom hydrogen, optionally substituted alkyl or optionally substitutedphenyl. In some such embodiments, R^(1e) is alkyl, preferably C₁₋₆alkyl, for example, methyl. In some other embodiments, R^(1e) ishydrogen. In some embodiments, R^(2e) is hydrogen. In some suchembodiments, R^(1f) is alkyl, preferably C₁₋₆ alkyl, for example,methyl. In some other embodiments, R^(1f) is hydrogen. In someembodiments, R^(2f) is hydrogen.

In some embodiments, the polymer may further comprise one or morerecurring units selected from the group consisting of polyacrylamides,polyacrylates, polyurethanes, polysiloxanes, silicones, polyacroleins,polyphosphazenes, polyisocyanates, poly-ols, and polysaccharides, orcombinations thereof. In some such embodiments, the polymer may furthercomprising one or more recurring units of polyacrylamide of Formula(IIIa) or (IIIb) with the structure shown above.

Some embodiments described herein are related to a grafted polymercomprising functionalized oligonucleotides covalently bonded to apolymer with a recurring unit of Formula (V) as described herein. Insome embodiments, the polymer may further comprise a recurring unit ofFormula (VIa) or (VIb), or both. In some embodiments, the covalentbonding between the functionalized oligonucleotide and the polymercomprises the structure moiety

or combinations thereof, wherein * indicates the polymer's point ofconnection with the functionalized oligonucleotide. In some suchembodiments, the covalent bonding between the functionalizedoligonucleotide and the polymer comprises structure moiety

In some embodiments, the grafted polymer is prepared by reacting one ormore functional moieties of the functionalized oligonucleotide with thepolymer, said one or more functional moieties comprise or are selectedfrom optionally substituted amino, hydroxy, thiol, carboxyl, acidanhydride, or combinations thereof. In one embodiment, thefunctionalized oligonucleotide comprises one or more optionallysubstituted amino groups. In some such embodiments, the amino group isunsubstituted. In some such embodiments, the grafted polymer is preparedby reacting the glycidyl ether or epoxy groups of the polymer with saidone or more amino groups of the functionalized oligonucleotides. In somesuch embodiments, the epoxy groups of the polymer are derived fromrecurring unit of Formula (VIa).

Substrates

Some embodiments described herein are related to a substrate having afirst surface comprising a polymer with a recurring unit of Formula (I)and a recurring unit of Formula (II) covalently bonded thereto asdescribed herein. In some embodiments, the polymer may further compriseone or more recurring units selected from the group consisting ofpolyacrylamides, polyacrylates, polyurethanes, polysiloxanes, silicones,polyacroleins, polyphosphazenes, polyisocyanates, poly-ols, andpolysaccharides, or combinations thereof. In some such embodiments, thepolymer may further comprising one or more recurring units ofpolyacrylamide of Formula (IIIa) or (IIIb) with the structure shownabove. In some embodiments, the covalent bonds between the polymer andthe substrate are formed by amine epoxy ring opening reaction. In someembodiments, the covalent bonding between the polymer and the firstsurface of the substrate comprises the structure moiety

or combinations thereof, wherein the substituted amino is derived fromthe recurring unit of Formula (II) and * indicates the polymer's pointof connection with the first surface of the substrate. In some suchembodiment, the covalent bonding between the polymer and the firstsurface comprises structure moiety

In some embodiments, the substrate is prepared by reacting the polymerwith a first plurality of functional groups covalently attached theretothe first surface, wherein the first plurality of functional groupscomprise or are selected from vinyl, acryloyl, alkenyl, cycloalkenyl,heterocycloalkenyl, alkynyl, cycloalkynyl, heterocycloalkynyl, nitrene,aldehyde, hydrazinyl, glycidyl ether, epoxy, carbene, isocyanate ormaleimide, or optionally substituted variants or combinations thereof.In some such embodiments, the first plurality of functional groupscomprises or are selected from epoxy groups. In one embodiment, saidepoxy group has the structure

In some embodiments, the substrate is prepared by reacting the aminogroups of the polymer with the epoxy groups of the first surface. Insome embodiments, the surface is pretreated with a functionalized silanecomprising said first plurality of the functional groups describedabove, and the polymer is covalently bonded to the first surface throughreaction with the first plurality of the functional groups of thefunctionalized silane.

In some embodiments, the substrate further comprises functionalizedoligonucleotides covalently bonded to the polymer. In some embodiment,the covalent bond between the oligonucleotide and the polymer is formedby azide click cycloaddition reaction. In some embodiments, the covalentbonding between the functionalized oligonucleotide and the polymercomprises the structure moiety

or combinations thereof, wherein * indicates the polymer's point ofconnection with the functionalized oligonucleotide. In some suchembodiments, the functionalized oligonucleotides are covalently bondedto the polymer by reacting one or more functional moieties of thefunctionalized oligonucleotides with the azido groups of the polymer,said one or more functional moieties comprise or are selected fromalkynes, cycloalkenes, cycloalkynes, heterocycloalkenes,heterocycloalkynes, or optionally substituted variants or combinationsthereof. In some such embodiments, said one or more functional moietiescomprise alkyne. In some other embodiments, said one or more functionalmoieties comprise or are selected from norbornene, cyclooctyne, orbicyclononyne, or optionally substituted variants or combinationsthereof. In one embodiment, the bicyclononyne isbicyclo[6.1.0]non-4-yne. In some embodiments, the functionalizedoligonucleotides are covalently bonded to the polymer by reacting theazido groups of the polymer with one or more alkyne moieties of thefunctionalized oligonucleotides. In some other embodiments, thefunctionalized oligonucleotides are covalently bonded to the polymer byreacting the azido groups of the polymer with one or morebicyclo[6.1.0]non-4-yne moieties of the functionalized oligonucleotides.

Some embodiments described herein are related to a substrate having afirst surface comprising a polymer with a recurring unit of Formula (IV)covalently bonded thereto as described herein. In some embodiments, thepolymer may further comprise one or more recurring units selected fromthe group consisting of polyacrylamides, polyacrylates, polyurethanes,polysiloxanes, silicones, polyacroleins, polyphosphazenes,polyisocyanates, poly-ols, and polysaccharides, or combinations thereof.In some such embodiments, the polymer may further comprising one or morerecurring units of polyacrylamide of Formula (IIIa) or (IIIb) with thestructure shown above. In some embodiments, the covalent bonds betweenthe polymer and the substrate are formed by tetrazine Diels-Alderreactions, which results in the elimination of nitrogen gas. In someembodiments, wherein the covalent bonding between the polymer and thefirst surface of the substrate comprises the structure moiety

or combinations thereof, wherein * indicates the polymer's point ofconnection with the first surface. In some such embodiments, thecovalent bonding between the polymer and the first surface comprisesstructure moiety

In some other such embodiments, the covalent bonding between the polymerand the first surface comprises structure moiety

In some embodiments, the substrate is prepared by reacting the polymerwith a first plurality of functional groups covalently attached theretothe first surface, wherein the first plurality of functional groupscomprise or are selected from vinyl, acryloyl, alkenyl, cycloalkenyl,heterocycloalkenyl, alkynyl, cycloalkynyl, heterocycloalkynyl, nitrene,aldehyde, hydrazinyl, glycidyl ether, epoxy, carbene, isocyanate ormaleimide, or optionally substituted variants or combinations thereof.In some such embodiments, the first plurality of functional groupscomprises, or are selected from, optionally substituted cycloalkenylgroups. In one embodiment, said cycloalkenyl is norbornene. In someembodiments, the substrate is prepared by reacting the tetrazine groupsof the polymer with the norbornene groups of the first surface. In someembodiments, the surface is pretreated with a functionalized silanecomprising said first plurality of the functional groups describedabove, and the polymer is covalently bonded to the first surface throughreaction with the first plurality of the functional groups of thefunctionalized silane.

In some embodiments, the substrate further comprises functionalizedoligonucleotides covalently bonded to the polymer. In some embodiments,the covalent bonding between the oligonucleotide and the polymer isformed by a tetrazine Diels-Alder reaction, which results in theelimination of nitrogen gas. In some embodiments, the covalent bondingbetween the functionalized oligonucleotide and the polymer comprises thestructure moiety

or combinations thereof, and wherein * indicates the polymer's point ofconnection with the functionalized oligonucleotide. In some suchembodiments, the covalent bonding between the functionalizedoligonucleotide and the polymer comprises structure moiety

In some such embodiments, the functionalized oligonucleotides arecovalently bonded to the polymer by reacting one or more functionalmoieties of the functionalized oligonucleotides with the tetrazinegroups of the polymer, said one or more functional moieties comprise orare selected from alkynes, cycloalkenes, cycloalkynes,heterocycloalkenes, heterocycloalkynes, or optionally substitutedvariants or combinations thereof. In some such embodiments, said one ormore functional moieties comprise or are selected from norbornene,cyclooctyne, or bicyclononyne, or optionally substituted variants orcombinations thereof. In one embodiment, the bicyclononyne isbicyclo[6.1.0]non-4-yne. In some such embodiments, the functionalizedoligonucleotides are covalently bonded to the polymer by reacting thetetrazine groups of the polymer with one or more bicyclo[6.1.0]non-4-ynemoieties of the functionalized oligonucleotides.

Some embodiments described herein are related to a substrate having afirst surface comprising a polymer with a recurring unit of Formula (V)covalently bonded thereto as described herein. In some embodiments, thepolymer may further comprise one or more recurring units selected fromthe group consisting of polyacrylamides, polyacrylates, polyurethanes,polysiloxanes, silicones, polyacroleins, polyphosphazenes,polyisocyanates, poly-ols, and polysaccharides, or combinations thereof.In some embodiments, the polymer may further comprise a recurring unitof Formula (VIa) or (VIb), or both. In some embodiments, the covalentbonds between the polymer and the substrate are formed by amine epoxyring opening reaction. In some other embodiments, the covalent bondsbetween the polymer and the substrate are formed by azide clickcycloaddition reaction. In some embodiments, the covalent bondingbetween the polymer and the first surface of the substrate comprises thestructure moiety

or combinations thereof, wherein * indicates the point of connection ofpolymer with the first surface. In some such embodiments, the covalentbonding between the polymer and the first surface comprises structuremoiety

In some other embodiments, the covalent bonding between the polymer andthe first surface comprises the structure moiety

or combinations thereof, wherein * indicates the point of connection ofpolymer with the first surface. In some such embodiments, the covalentbonding between the polymer and the first surface comprises structuremoiety

In some other such embodiments, the covalent bonding between the polymerand the first surface comprises structure moiety

In some embodiments, the substrate is prepared by reacting the polymerwith a first plurality of functional groups covalently attached theretothe first surface, wherein the first plurality of functional groupscomprise or are selected from vinyl, acryloyl, alkenyl, cycloalkenyl,heterocycloalkenyl, alkynyl, cycloalkynyl, heterocycloalkynyl, nitrene,aldehyde, hydrazinyl, glycidyl ether, epoxy, carbene, isocyanate ormaleimide, or optionally substituted variants or combinations thereof.In some such embodiments, the first plurality of functional groupscomprises or are selected from optionally substituted cycloalkenylgroups. In one embodiment, said cycloalkenyl is norbornene. In someembodiments, the substrate is prepared by reacting the azido groups ofthe polymer with the norbornene groups of the first surface. In someother embodiments, the first plurality of functional groups comprises orare selected from glycidyl ether or epoxy groups. In some embodiments,the substrate is prepared by deprotecting the Boc-protected amino of thepolymer and then reacting the amino groups of the polymer with theglycidyl ether or epoxy groups of the first surface. In someembodiments, the surface is pretreated with a functionalized silanecomprising said first plurality of the functional groups describedabove, and the polymer is covalently bonded to the first surface throughreaction with the first plurality of the functional groups of thefunctionalized silane.

In some embodiments, the substrate further comprises functionalizedoligonucleotides covalently bonded to the polymer. In some embodiments,the covalent bonding between the oligonucleotide and the polymer isformed by amine epoxy ring opening reaction. In some embodiments, thecovalent bonding between the functionalized oligonucleotide and thepolymer comprises the structure moiety

or combinations thereof, wherein * indicates the point of connection ofpolymer with the functionalized oligonucleotide. In some embodiments,the covalent bonding between the functionalized oligonucleotide and thepolymer comprises structure moiety

In some such embodiments, the functionalized oligonucleotides arecovalently bonded to the polymer by reacting one or more functionalmoieties of the functionalized oligonucleotides with the epoxy groups ofthe polymer, said one or more functional moieties comprise or areselected from optionally substituted amino, hydroxy, thiol, carboxyl,acid anhydride, or combinations thereof. In some such embodiments, saidone or more functional moieties comprise or are selected from optionallysubstituted amino groups. In some such embodiments, the functionalizedoligonucleotides are covalently bonded to the polymer by reacting theepoxy groups of the polymer with the amino groups of the functionalizedoligonucleotides.

In embodiments described herein, the substrate material may compriseglass, silica, plastic, quartz, metal, metal oxide, or combinationsthereof. In some embodiments, the substrate comprises glass. In someembodiments, the first surface of the substrate comprises both polymercoated regions and inert regions.

Substrate Surface Preparations

Some embodiments described herein are related to processes or methodsfor immobilizing a grafted polymer to a first surface of a substrate byproviding a substrate having a first surface having a first plurality offunctional groups covalently attached thereto; providing a graftedpolymer having functionalized oligonucleotides covalently bonded to apolymer, wherein the polymer comprises a second plurality of functionalgroups; and reacting the first plurality functional groups of the firstsurface with the second plurality of functional groups of the polymersuch that the polymer is covalently bonded to the first surface of thesubstrate.

In some embodiments of the methods described herein, the first surfaceof the substrate is pretreated with a functionalized silane, whereinsaid functionalized silane comprises the first plurality of thefunctional groups. In some embodiments, the functionalized silane isdeposited onto the surface by Chemical Vapor Deposition (CVD) method. Insome such embodiments, functionalized silane can be applied onto thefirst surface by CVD method using Yield Engineering Systems (YES) oven.

In some embodiments of the methods described herein, the grafted polymeris formed by reacting a third plurality of functional groups of thepolymer with one or more functional moieties of the functionalizedoligonucleotides. In some other embodiments, the grafted polymer isformed by reacting said one or more functional moieties offunctionalized oligonucleotides with monomers comprising a thirdplurality of functional groups; polymerizing the reacted monomers toform the polymer such that the functionalized oligonucleotides arecovalently bonded to the polymer.

In some embodiments of the methods described herein, the secondplurality of functional groups of the polymer are the same as the thirdplurality of functional groups of the polymer. For example, the secondplurality and the third plurality of functional groups of the polymercan both be tetrazines. In some other embodiments, the functional groupsin the second plurality of functional groups of the polymer aredifferent from the functional groups in the third plurality offunctional groups of the polymer.

The polymer backbone used in the methods described herein can be linear,branched, hyper-branched or dendritic. The final polymer structure canbe in different architectures, including, for example, random copolymer,block copolymer, comb-shaped polymer or star-shaped polymerarchitectures. Different classes of polymer backbones can be used in themethods described herein, including but not limited to polyacrylamides,polyacrylates, polyurethanes, polysiloxanes, silicones, polyacroleins,polyphosphazenes, polyisocyanates, poly-ols, polysaccharides, etc. Insome embodiments, the polymer comprises polyacrylamide backbone. In someother embodiments, the polymer comprises polyacrylate backbone. In stillsome other embodiments, the polymer comprises polyurethane backbone. Instill some other embodiments, the polymer comprises polyphosphazenesbackbone. In still some other embodiments, the polymer comprises adendrimer backbone.

In some embodiments of the methods described herein, the first pluralityof functional groups of the first surface comprise or are selected fromvinyl, acryloyl, alkenyl, cycloalkenyl, heterocycloalkenyl, alkynyl,cycloalkynyl, heterocycloalkynyl, nitrene, aldehyde, hydrazinyl,glycidyl ether, epoxy, carbene, isocyanate or maleimide, or optionallysubstituted variants or combinations thereof. In some such embodiments,the first plurality of functional groups comprises or is selected fromcycloalkenyl, glycidyl ether, epoxy, or optionally substituted variantsor combinations thereof. In some further embodiments, the firstplurality of functional groups comprises or is selected from norbornene.In some other embodiments, the first plurality of functional groupscomprises an epoxy. In still some other embodiments, the first pluralityof functional groups comprises glycidyl ether.

In some embodiments of the method described herein, the functionalgroups of the polymer may comprise or are selected from amino,tetrazinyl, azido, carboxyl, hydroxy, thiol, aldehyde, halo, alkenyl,alkynyl, epoxy, glycidyl ether, etc. In some embodiments of the methodsdescribed herein, the second plurality of functional groups of thepolymer comprise or are selected from amino, tetrazinyl, azido,carboxyl, hydroxy, thiol, aldehyde, or optionally substituted variantsor combinations thereof. In some such embodiments, the second pluralityof functional groups comprise or are selected from amino or protectedamino, for example, Boc-protected amino. In some other embodiments, thesecond plurality of functional groups comprises optionally substitutedtetrazinyl. In still some other embodiments, the second plurality offunctional groups comprises azido.

In some embodiments of the methods described herein, the third pluralityof functional groups of the polymer comprises or is selected from azido,tetrazinyl, glycidyl, epoxy, alkynyl, or optionally substituted variantsor combinations thereof. In some such embodiments, the third pluralityof functional groups comprises azido. In some other embodiments, thethird plurality of functional groups comprises optionally substitutedtetrazinyl. In yet some other embodiments, the third plurality offunctional groups comprises alkynyl. In yet some other embodiments, thethird plurality of functional groups comprises optionally substitutedglycidyl ether.

In some embodiments of the methods described herein, said one or morefunctional moieties of the functionalized oligonucleotides comprise orare selected from amino, azido, carboxyl, acid anhydride, tetrazine,epoxy, glycidyl ether, vinyl, acryloyl, alkenyl, cycloalkenyl, alkynyl,cycloalkynyl, nitrene, aldehyde, hydrazinyl, or maleimide or optionallysubstituted variants or combinations thereof. In some furtherembodiments, said one or more functional moieties of the functionalizedoligonucleotides comprise or are selected from alkynyl, cycloalkenyl,cycloalkynyl, amino, azido, hydroxy, thiol, carboxyl, acid anhydride, oroptionally substituted variants or combinations thereof. In some suchembodiments, said one or more functional moieties comprise cycloalkynyl,for example, bicyclo[6.1.0] non-4-yne (BCN). In some other embodiments,said one or more functional moieties comprise alkynyl. In still someother embodiments, said one or more functional moieties comprise azido.In still some other embodiments, said one or more functional moietiescomprise optionally substituted amino.

In some embodiments of the methods described herein, the grafted polymercomprises functionalized oligonucleotides covalently bonded to a polymerwith a recurring unit of Formula (I) and a recurring unit of Formula(II) as described herein. In some such embodiments, the first pluralityof functional groups of the first surface comprise epoxy groups. In oneembodiment, said epoxy group is

In some such embodiments, the grafted polymer is covalently bonded tothe first surface by reacting the amino groups of the polymer with theepoxy groups of the first surface.

In some embodiments of the methods described herein, the grafted polymercomprises functionalized oligonucleotides covalently bonded to a polymerwith a recurring unit of Formula (IV) as described herein. In some suchembodiments, the first plurality of functional groups of the firstsurface comprises optionally substituted cycloalkenyl groups, forexample, optionally substituted norbornene. In some such embodiments,the grafted polymer is covalently bonded to the first surface byreacting the tetrazine groups of the polymer with the norbornene groupsof the first surface.

In some embodiments of the methods described herein, the grafted polymercomprises functionalized oligonucleotides covalently bonded to a polymerwith a recurring unit of Formula (V) as described herein. In someembodiments, the polymer may further comprise a recurring unit ofFormula (VIa) or (VIb), or both, as described herein. In some suchembodiments, the first plurality of functional groups comprisesoptionally substituted cycloalkenyl groups, for example, optionallysubstituted norbornene. In some such embodiments, the grafted polymer iscovalently bonded to the first surface by reacting the azido groups ofthe polymer with the norbornene groups of the first surface. In someother embodiments, the first plurality of functional groups comprisesglycidyl ether or epoxy groups. In some other embodiments, the graftedpolymer is covalently bonded to the first surface by deprotecting theBoc-protected amino groups of the polymer; and reacting the amino groupsof the polymer with the glycidyl ether or epoxy groups of the firstsurface.

Some embodiments described herein are related to processes or methodsfor immobilizing a polymer described herein to a first surface of asubstrate, comprising: providing a substrate having a first surfacecomprising a first plurality of functional groups covalently attachedthereto; providing a polymer with recurring units of Formulae (I) and(II), Formula (IV), or Formula (V) as described herein; and reacting thefirst plurality functional groups of the first surface with the polymersuch that the polymer is covalently bonded to the first surface of thesubstrate. In some such embodiments, the processes or methods furthercomprises providing functionalized oligonucleotides comprising one ormore functionalized moieties; and reacting said one or morefunctionalized moieties with the polymer such that the functionalizedoligonucleotides are covalently bonded to the polymer. In some suchembodiments, the first plurality of functional groups of the firstsurface comprises or are selected from vinyl, acryloyl, alkenyl,cycloalkenyl, heterocycloalkenyl, alkynyl, cycloalkynyl,heterocycloalkynyl, nitrene, aldehyde, hydrazinyl, glycidyl ether,epoxy, carbene, isocyanate or maleimide, or optionally substitutedvariants or combinations thereof. In some such embodiments, said one ormore functionalized moieties comprise or are selected from amino, azido,carboxyl, acid anhydride, tetrazine, epoxy, glycidyl ether, vinyl,acryloyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, nitrene,aldehyde, hydrazinyl, or maleimide or optionally substituted variants orcombinations thereof.

In any embodiments of the methods described herein, the polymer orgrafted polymer with recurring units of Formulae (I) and (II), Formula(IV), or Formula (V) may further comprise one or more recurring unitsselected from the group consisting of polyacrylamides, polyacrylates,polyurethanes, polysiloxanes, silicones, polyacroleins,polyphosphazenes, polyisocyanates, poly-ols, and polysaccharides, orcombinations thereof. In some such embodiments, the polymer may furthercomprise one or more recurring units of polyacrylamide of Formula (IIIa)or (IIIb) with the structure shown above.

In any embodiments of the methods described herein, the method furthercomprises a washing step to remove excess unbounded functionalizedoligonucleotides. In some embodiments, the method further comprises adrying step.

In any of the embodiments described herein, the substrate can comprise amaterial selected from glass, silica, quartz, plastic, metal, metaloxide, patterned or not or combinations thereof. In one embodiment, thesurface of the substrate comprises glass. In some embodiments, thesurface of the substrate can comprise both functionalized silane coatedregions and inert regions. In some embodiments, the inert regions areselected from glass regions, metal regions, mask regions andinterstitial regions, or combinations thereof. In one embodiment, theinert regions comprise glass.

In any of the embodiments described herein, QC markers can be includedin the polymer and/or primer structures.

In any embodiments described herein, the polymer or grafted polymer maybe applied to the surface of the substrate via various surfaceapplication techniques known to one skilled in the art, for example,spin coating, spray coating, dip coating, ink-jet coating, etc.

Substrates Materials and Design

In some embodiments, substrates used in the present application includesilica-based substrates, such as glass, fused silica and othersilica-containing materials. In some embodiments, silica-basedsubstrates can also be silicon, silicon dioxide, silicon nitride,silicone hydrides. In some embodiments, substrates used in the presentapplication include plastic materials such as polyethylene, polystyrene,poly(vinyl chloride), polypropylene, nylons, polyesters, polycarbonatesand poly(methyl methacrylate). Preferred plastics material ispoly(methyl methacrylate), polystyrene and cyclic olefin polymersubstrates. In some embodiments, the substrate is a silica-basedmaterial or plastic material. In particular embodiments, the substratehas at least one surface comprising glass.

In some embodiments, the substrates can be, or can contain, a metal. Insome such embodiments, the metal is gold. In some embodiments, thesubstrate has at least one surface comprising a metal oxide. In oneembodiment, the surface comprises a tantalum oxide or tin oxide.

Acrylamide, enone, or acrylate may also be utilized as a substratematerial. Other substrate materials can include, but are not limited togallium arsenide, indium phosphide, aluminum, ceramics, polyimide,quartz, resins, polymers and copolymers. The foregoing lists areintended to be illustrative of, but not limiting to the presentapplication.

In some embodiments, the substrate and/or the substrate surface can bequartz. In some other embodiments, the substrate and/or the substratesurface can be semiconductor, i.e. GaAs or ITO.

Substrates can comprise a single material or a plurality of differentmaterials. Substrates can be composites or laminates. Substrate can beflat, round, textured and patterned. Patterns can be formed, forexample, by metal pads that form features on non-metallic surfaces, forexample, as described in U.S. Pat. No. 8,778,849, which is incorporatedherein by reference. Another useful patterned surface is one having wellfeatures formed on a surface, for example, as described in U.S. Pat.App. Pub. No. 2014/0243224 A1, U.S. Pat. App. Pub. No. 2011/0172118 A1or U.S. Pat. No. 7,622,294, each of which is incorporated herein byreference in its entirety. For embodiments that use a patternedsubstrate, a gel can be selectively attached to the pattern features(e.g. gel can be attached to metal pads or gel can be attached to theinterior of wells) or alternatively the gel can be uniformly attachedacross both the pattern features and the interstitial regions.

Advantages in using plastics-based substrates in the preparation and useof molecular arrays include cost: the preparation of appropriateplastics-based substrates by, for example injection-molding, isgenerally cheaper than the preparation, e.g. by etching and bonding, ofsilica-based substrates. Another advantage is the nearly limitlessvariety of plastics allowing fine-tuning of the optical properties ofthe support to suit the application for which it is intended or to whichit may be put.

Where metals are used as substrates or as pads on a substrate, this maybe because of the desired application: the conductivity of metals canallow modulation of the electric field in DNA-based sensors. In thisway, DNA mismatch discrimination may be enhanced, the orientation ofimmobilized oligonucleotide molecules can be affected, or DNAhybridization kinetics can be accelerated.

In some embodiments, the substrate is silica-based but the shape of thesubstrate employed may be varied in accordance with the application forwhich the present application is practiced. Generally, however, slidesof support material, such as silica, e.g. fused silica, are ofparticular utility in the preparation and subsequent integration ofmolecules. Of particular use in the practice of the present applicationare fused silica slides sold under the trade name SPECTROSIL™. Thisnotwithstanding, it will be evident to the skilled person that thepresent application is equally applicable to other presentations ofsubstrate (including silica-based supports), such as beads, rods and thelike.

In some embodiments, the surface of the substrate comprises bothfunctional molecules-coated regions and inert regions with no coatings.In some such embodiments, the functionalized molecule coatings arehydrogel or polymer coatings. The functional molecules-coated regionscan comprise reactive sites, and thus, can be used to attach moleculesthrough chemical bonding or other molecular interactions. In someembodiments, the functional molecules-coated regions (e.g. reactivefeatures, pads, beads, posts or wells) and the inert regions (referredto as interstitial regions) can alternate so as to form a pattern or agrid. Such patterns can be in one or two dimensions. In someembodiments, the inert regions can be selected from glass regions, metalregions, mask regions, or combinations thereof. Alternatively thesematerials can form reactive regions. Inertness or reactivity will dependon the chemistry and processes used on the substrate. In one embodiment,the surface comprises glass regions. In another embodiment, the surfacecomprises metal regions. In still another embodiment, the surfacecomprises mask regions. In some embodiments of the compositionsdescribed herein, the substrate can be a bead. Non-limiting exemplarysubstrate materials that can be coated with a polymer of the presentdisclosure or that can otherwise be used in a composition or method setforth herein are described in U.S. Pat. Nos. 8,778,848 and 8,778,849,each of which is incorporated herein by reference.

In some embodiments, a substrate described herein forms at least part ofa flow cell or is located in a flow cell. In some such embodiments, theflow cells further comprise polynucleotides attached to the surface ofthe substrate via the functional molecules coating, for example, apolymer coating. In some embodiments, the polynucleotides are present inthe flow cells in polynucleotide clusters, wherein the polynucleotidesof the polynucleotide clusters are attached to a surface of the flowcell via the polymer coating. In such embodiments, the surface of theflow cell body to which the polynucleotides are attached is consideredthe substrate. In other embodiments, a separate substrate having apolymer coated surface is inserted into the body of the flow cell. Inpreferred embodiments, the flow cell is a flow chamber that is dividedinto a plurality of lanes or a plurality of sectors, wherein one or moreof the plurality of lanes or plurality of sectors comprises a surfacethat is coated with a covalently attached polymer coating describedherein. In some embodiments of the flow cells described herein, theattached polynucleotides within a single polynucleotide cluster have thesame or similar nucleotide sequence. In some embodiments of the flowcells described herein, the attached polynucleotides of differentpolynucleotide clusters have different or nonsimilar nucleotidesequences. Exemplary flow cells and substrates for manufacture of flowcells that can be used in method or composition set forth hereininclude, but are not limited to, those commercially available fromIllumina, Inc. (San Diego, Calif.) or described in US 2010/0111768 A1 orUS 2012/0270305, each of which is incorporated herein by reference.

Sequencing Application

A composition, apparatus or method set forth herein can be used with anyof a variety of amplification techniques. Exemplary techniques that canbe used include, but are not limited to, polymerase chain reaction(PCR), rolling circle amplification (RCA), multiple displacementamplification (MDA), or random prime amplification (RPA). In particularembodiments, one or more primers used for amplification can be attachedto a polymer coating. In PCR embodiments, one or both of the primersused for amplification can be attached to a polymer coating. Formatsthat utilize two species of attached primer are often referred to asbridge amplification because double stranded amplicons form abridge-like structure between the two attached primers that flank thetemplate sequence that has been copied. Exemplary reagents andconditions that can be used for bridge amplification are described, forexample, in U.S. Pat. No. 5,641,658; U.S. Patent Publ. No. 2002/0055100;U.S. Pat. No. 7,115,400; U.S. Patent Publ. No. 2004/0096853; U.S. PatentPubl. No. 2004/0002090; U.S. Patent Publ. No. 2007/0128624; and U.S.Patent Publ. No. 2008/0009420, each of which is incorporated herein byreference in its entirety. PCR amplification can also be carried outwith one of the amplification primers attached to a polymer coating andthe second primer in solution. An exemplary format that uses acombination of one attached primer and soluble primer is emulsion PCR asdescribed, for example, in Dressman et al., Proc. Natl. Acad. Sci. USA100:8817-8822 (2003), WO 05/010145, or U.S. Patent Publ. Nos.2005/0130173 or 2005/0064460, each of which is incorporated herein byreference. Emulsion PCR is illustrative of the format and it will beunderstood that for purposes of the methods set forth herein the use ofan emulsion is optional and indeed for several embodiments an emulsionis not used. Furthermore, primers need not be attached directly tosubstrate or solid supports as set forth in the ePCR references and caninstead be attached to a polymer coating as set forth herein.

RCA techniques can be modified for use with a method, composition orapparatus of the present disclosure. Exemplary components that can beused in an RCA reaction and principles by which RCA produces ampliconsare described, for example, in Lizardi et al., Nat. Genet. 19:225-232(1998) and US 2007/0099208 A1, each of which is incorporated herein byreference. Primers used for RCA can be in solution or attached to apolymer coating.

MDA techniques can be modified for use with a method, composition orapparatus of the present disclosure. Some basic principles and usefulconditions for MDA are described, for example, in Dean et al., Proc.Natl. Acad. Sci. USA 99:5261-66 (2002); Lage et al., Genome Research13:294-307 (2003); Walker et al., Molecular Methods for Virus Detection,Academic Press, Inc., 1995; Walker et al., Nucl. Acids Res. 20:1691-96(1992); U.S. Pat. Nos. 5,455,166; 5,130,238; and 6,214,587, each ofwhich is incorporated herein by reference. Primers used for MDA can bein solution or attached to a polymer coating.

In particular embodiments a combination of the above-exemplifiedamplification techniques can be used. For example, RCA and MDA can beused in a combination wherein RCA is used to generate a concatemericamplicon in solution (e.g. using solution-phase primers). The ampliconcan then be used as a template for MDA using primers that are attachedto a polymer coating. In this example, amplicons produced after thecombined RCA and MDA steps will be attached to the polymer coating.

In some embodiments, the functionalized hydrogel or polymer-coatedsubstrate described herein can be used in a method for determining anucleotide sequence of a polynucleotide. In such embodiments, the methodcan comprise the steps of (a) contacting a polynucleotide polymerasewith polynucleotide clusters attached to a surface of a substrate viaany one of the polymer or hydrogel coatings described herein; (b)providing nucleotides to the polymer-coated surface of the substratesuch that a detectable signal is generated when one or more nucleotidesare utilized by the polynucleotide polymerase; (c) detecting signals atone or more polynucleotide clusters; and (d) repeating steps (b) and(c), thereby determining a nucleotide sequence of a polynucleotidepresent at the one or more polynucleotide clusters.

Nucleic acid sequencing can be used to determine a nucleotide sequenceof a polynucleotide by various processes known in the art. In apreferred method, sequencing-by-synthesis (SBS) is utilized to determinea nucleotide sequence of a polynucleotide attached to a surface of asubstrate via any one of the polymer coatings described herein. In suchprocess, one or more nucleotides are provided to a templatepolynucleotide that is associated with a polynucleotide polymerase. Thepolynucleotide polymerase incorporates the one or more nucleotides intoa newly synthesized nucleic acid strand that is complementary to thepolynucleotide template. The synthesis is initiated from anoligonucleotide primer that is complementary to a portion of thetemplate polynucleotide or to a portion of a universal or non-variablenucleic acid that is covalently bound at one end of the templatepolynucleotide. As nucleotides are incorporated against the templatepolynucleotide, a detectable signal is generated that allows for thedetermination of which nucleotide has been incorporated during each stepof the sequencing process. In this way, the sequence of a nucleic acidcomplementary to at least a portion of the template polynucleotide canbe generated, thereby permitting determination of the nucleotidesequence of at least a portion of the template polynucleotide.

Flow cells provide a convenient format for housing an array that isproduced by the methods of the present disclosure and that is subjectedto a sequencing-by-synthesis (SBS) or other detection technique thatinvolves repeated delivery of reagents in cycles. For example, toinitiate a first SBS cycle, one or more labeled nucleotides, DNApolymerase, etc., can be flowed into/through a flow cell that houses anucleic acid array made by methods set forth herein. Those sites of anarray where primer extension causes a labeled nucleotide to beincorporated can be detected. Optionally, the nucleotides can furtherinclude a reversible termination property that terminates further primerextension once a nucleotide has been added to a primer. For example, anucleotide analog having a reversible terminator moiety can be added toa primer such that subsequent extension cannot occur until a deblockingagent is delivered to remove the moiety. Thus, for embodiments that usereversible termination, a deblocking reagent can be delivered to theflow cell (before or after detection occurs). Washes can be carried outbetween the various delivery steps. The cycle can then be repeated ntimes to extend the primer by n nucleotides, thereby detecting asequence of length n. Exemplary SBS procedures, fluidic systems anddetection platforms that can be readily adapted for use with an arrayproduced by the methods of the present disclosure are described, forexample, in Bentley et al., Nature 456:53-59 (2008), WO 04/018497; U.S.Pat. No. 7,057,026; WO 91/06678; WO 07/123744; U.S. Pat. Nos. 7,329,492;7,211,414; 7,315,019; 7,405,281, and US 2008/0108082, each of which isincorporated herein by reference in its entirety.

Other sequencing procedures, including for example those that use cyclicreactions, can be used, such as pyrosequencing. Pyrosequencing detectsthe release of inorganic pyrophosphate (PPi) as particular nucleotidesare incorporated into a nascent nucleic acid strand (Ronaghi, et al.,Analytical Biochemistry 242(1), 84-9 (1996); Ronaghi, Genome Res. 11(1),3-11 (2001); Ronaghi et al. Science 281(5375), 363 (1998); U.S. Pat.Nos. 6,210,891; 6,258,568 and 6,274,320, each of which is incorporatedherein by reference in its entirety). In pyrosequencing, released PPican be detected by being immediately converted to adenosine triphosphate(ATP) by ATP sulfurylase, and the level of ATP generated can be detectedvia luciferase-produced photons. Thus, the sequencing reaction can bemonitored via a luminescence detection system. Excitation radiationsources used for fluorescence based detection systems are not necessaryfor pyrosequencing procedures. Useful fluidic systems, detectors andprocedures that can be used for application of pyrosequencing to arraysof the present disclosure are described, for example, in WO 12/058096A1, US 2005/0191698 A1, U.S. Pat. Nos. 7,595,883, and 7,244,559, each ofwhich is incorporated herein by reference in its entirety.

Sequencing-by-ligation reactions are also useful including, for example,those described in Shendure et al. Science 309:1728-1732 (2005); U.S.Pat. Nos. 5,599,675; and 5,750,341, each of which is incorporated hereinby reference in its entirety. Some embodiments can includesequencing-by-hybridization procedures as described, for example, inBains et al., Journal of Theoretical Biology 135(3), 303-7 (1988);Drmanac et al., Nature Biotechnology 16, 54-58 (1998); Fodor et al.,Science 251(4995), 767-773 (1995); and WO 1989/10977, each of which isincorporated herein by reference in its entirety. In bothsequencing-by-ligation and sequencing-by-hybridization procedures,nucleic acids that are present at sites of an array are subjected torepeated cycles of oligonucleotide delivery and detection. Fluidicsystems for SBS methods as set forth herein or in references citedherein can be readily adapted for delivery of reagents forsequencing-by-ligation or sequencing-by-hybridization procedures.Typically, the oligonucleotides are fluorescently labeled and can bedetected using fluorescence detectors similar to those described withregard to SBS procedures herein or in references cited herein.

Some embodiments can utilize methods involving the real-time monitoringof DNA polymerase activity. For example, nucleotide incorporations canbe detected through fluorescence resonance energy transfer (FRET)interactions between a fluorophore-bearing polymerase andγ-phosphate-labeled nucleotides, or with zeromode waveguides (ZMWs).Techniques and reagents for FRET-based sequencing are described, forexample, in Levene et al. Science 299, 682-686 (2003); Lundquist et al.Opt. Lett. 33, 1026-1028 (2008); Korlach et al. Proc. Natl. Acad. Sci.USA 105, 1176-1181 (2008), the disclosures of which are incorporatedherein by reference in its entirety.

Some SBS embodiments include detection of a proton released uponincorporation of a nucleotide into an extension product. For example,sequencing based on detection of released protons can use an electricaldetector and associated techniques that are commercially available fromIon Torrent (Guilford, CT, a Life Technologies subsidiary) or sequencingmethods and systems described in US 2009/0026082 A1; US 2009/0127589 A1;US 2010/0137143 A1; or US 2010/0282617 A1, each of which is incorporatedherein by reference in its entirety.

Another useful application for an array of the present disclosure, forexample, having been produced by a method set forth herein, is geneexpression analysis. Gene expression can be detected or quantified usingRNA sequencing techniques, such as those, referred to as digital RNAsequencing. RNA sequencing techniques can be carried out usingsequencing methodologies known in the art such as those set forth above.Gene expression can also be detected or quantified using hybridizationtechniques carried out by direct hybridization to an array or using amultiplex assay, the products of which are detected on an array. Anarray of the present disclosure, for example, having been produced by amethod set forth herein, can also be used to determine genotypes for agenomic DNA sample from one or more individual. Exemplary methods forarray-based expression and genotyping analysis that can be carried outon an array of the present disclosure are described in U.S. Pat. Nos.7,582,420; 6,890,741; 6,913,884 or 6,355,431 or U.S. Pat. Pub. Nos.2005/0053980 A1; 2009/0186349 A1 or US 2005/0181440 A1, each of which isincorporated herein by reference in its entirety.

In some embodiments of the above-described method which employ a flowcell, only a single type of nucleotide is present in the flow cellduring a single flow step. In such embodiments, the nucleotide can beselected from the group consisting of dATP, dCTP, dGTP, dTTP and analogsthereof. In other embodiments of the above-described method which employa flow cell, a plurality of different types of nucleotides are presentin the flow cell during a single flow step. In such methods, thenucleotides can be selected from dATP, dCTP, dGTP, dTTP and analogsthereof.

Determination of the nucleotide or nucleotides incorporated during eachflow step for one or more of the polynucleotides attached to the polymercoating on the surface of the substrate present in the flow cell isachieved by detecting a signal produced at or near the polynucleotidetemplate. In some embodiments of the above-described methods, thedetectable signal comprises and optical signal. In other embodiments,the detectable signal comprises a non-optical signal. In suchembodiments, the non-optical signal comprises a change in pH at or nearone or more of the polynucleotide templates.

EXAMPLES

Additional embodiments are disclosed in further detail in the followingexamples, which are not in any way intended to limit the scope of theclaims.

Example 1

Scheme 1 illustrates a synthetic scheme for the preparation ofpoly-acrylamide-co-AzAPA-co-aminoethylacrylamide (L-PAmAzA). In thefirst step, N-(5-azidoacetamidylpentyl) acrylamide (AzAPA) wassynthesized from reacting N-(5-bromoacetamidylpentyl) acrylamide (BrAPA)with sodium azide in DMF at 35° C. for 2 hours. Then, L-PAmAzA wassynthesized using an AIBN-type polymer initiation system (Vazo56) byreacting AzAPA with acrylamide and N-(2-aminoethyl)methacrylamide HCl.The resulting L-PAmAzA has the three recurring units in the molar ratiox:y:z of about 90% to about 5% to about 5%.

In addition, crosslinking of the L-PAmAzA was achieved by theintroduction of N,N′-methylenebisacrylamide monomers into thepolymerization reaction, which resulted in a crosslinked polymer(XL-PAmAzA) with exemplary structure shown as follows:

A series of linear polyacrylamides bearing orthogonal functionalitieswere prepared using the thermal initiator Vazo56 following the similarprocedure described above. The reaction time was about 1.5 hours toabout 3 hours, followed by a purification step through precipitationinto MeCN. Table 1 below summarizes the amounts of the monomers for thepolymerization reactions.

TABLE 1 Polymer M1 (mol %) M2 (mol %) M3a/b (mol %) M4 (mol %) 1 5 5 a:90 0 2 5 5 a: 90 1 3 5 10 a: 85 1 4 5 5 b: 90 1 5 5 5 a: 90 2 6 5 5 a:90 0.5

In order to demonstrate the orthogonal reactivity of the bifunctionalpolyacrylamides, the coating performance of three new polyacrylamides inTable 1 (Polymer 1, “P1”; Polymer 4, “P4”; Polymer 6, “P6”) containing 5mol % aminoethyl functionality on epoxy monolayer surface was assessedagainst the standard norbornene monolayer surface. Polymer 1 and Polymer6 are L-PAmAzA and XL-PAmAzA with the structures illustrated above. Thesimplified structure of Polymer 4 is shown below.

Standard PAZAM polymer was used as control. The flow cell layout for thenorbornene monolayer surface and the epoxy monolayer surface aresummarized in Table 2 and Table 3 respectively.

TABLE 2 Polymer Polymer cou- Vol. coupling pling std. [Final [P5/ Chan-Tempera- time PAZAM PAZAM]/ P7]/ nel ture (° C.) (min) (uL) Polymer w/v% uM 1 60 60 420 PAZAM 0.5 18 control 2 60 60 420 P1 0.5 18 3 60 60 420P4 0.5 18 4 60 60 420 P6 0.5 18 5 60 60 420 PAZAM 0.5 18 control 6 60 60420 P1 0.5 18 7 60 60 420 P4 0.5 18 8 60 60 420 P6 0.5 18

TABLE 3 Polymer Polymer cou- Vol. coupling pling std. [Final [P5/ Chan-tempera- time PAZAM PAZAM]/ P7]/ nel ture (° C.) (min) (uL) Polymer w/v% uM 1 60 60 420 PAZAM 0.5 18 control 2 60 60 420 P1 0.5 18 3 60 60 420P4 0.5 18 4 60 60 420 P6 0.5 18 5 60 60 420 PAZAM 0.5 18 control 6 60 60420 P1 0.5 18 7 60 60 420 P4 0.5 18 8 60 60 420 P6 0.5 18

HiSeq substrates (provided by ILLUMINA, San Diego, Calif.) were used forthis initial screening and the CVD process was performed using adesiccator. The bifunctional polyacrylamides polymers reacted withnorbornene via strain-promoted azide click reaction to covalently bondto the norbornene monolayer surface at 60° C. Similarly, bifunctionalpolyacrylamides polymers were coated onto the epoxy monolayers via epoxyring opening reaction with amine functional groups which results incovalent bonding of the polymers to the surface. Two QC metrics wereused to measure the success of the method. Both QC1 and QC3 utilizegreen laser, with PMT at 450V and filter emission at 555BP. The TET QColigo mix for QC1 is 1.6 mM: 100 mL oligos at 16 μM+0.9 mL HT1. The TETQC oligo mix for QC3 is 0.6 mM (each): 35 mL oligos at 16 μM+0.9mL HT1.The Typhoon florescence image of the polymers coated flow cell and therelated chart of median Typhoon intensity of the polymers on thenorbornene silane monolayer surface for TET QC1 and TET QC3 areillustrated in FIGS. 1A, 1B, 1C and 1D respectively. The Typhoonflorescence image of the polymers coated flow cell and the related chartof median Typhoon intensity of the polymers on the epoxy silanemonolayer surface for TET QC1 and TET QC3 are illustrated in FIGS. 2A,2B, 2C and 2D respectively.

TET QC measurements for the norbornene surface and the epoxy surface aresummarized in Table 4 and Table 5 respectively.

TABLE 4 % Intensity change, % Surface Lanes QC1->QC3 Loss Polymer 1 11%−11% PAZAM control 2 −1% 1% P1 3 0% 0% P4 4 −3% 3% P6 5 13% −13% PAZAMcontrol 6 −2% 2% P1 7 −6% 6% P4 8 −5% 5% P6

TABLE 5 % Intensity change, % Surface Lanes QC1->QC3 Loss Polymer 1 84%−84% PAZAM control 2 31% −31% P1 3 11% −11% P4 4 13% −13% P6 5 84% −84%PAZAM control 6 32% −32% P1 7 12% −12% P4 8 24% −24% XL-PAAm3

The results from the above noted pair of flow cells provided evidencesupporting the use of orthogonal reactivity of the polyacrylamidematerials to support Sequencing-by-Synthesis. First, all theazido-functionalized materials tested were capable of adhering securelyto a norbornene surface. This means that the azide incorporation intothe polymer structure was such that a stable surface could be obtained,as measured by a thermal stress test. Second, all of theamine-functionalized polymers were capable of coating the epoxy surfacethat was generated by the use of a desiccator. The surface primerdensities were approximately 20-30 k. In these experiments, the controlpolymer (i.e., the standard PAZAM), which contained no aminefunctionality, showed the largest surface loss after the thermal stresstest. This is the expected result. The bifunctionalized polyacrylamidepolymers P1, P4 and P6, each with 5% amine functionality, showedreasonable surface stability. The results indicated that thesepolyacrylamide coated surfaces were robust (surface losses ranging fromabout 20% to 30% after subjecting the polymer coated surface to thestandard Stress test). The results of TET QC signal changes of thenorbornene monolayer surface and the epoxy monolayer surface are shownin FIG. 3A and FIG. 3B respectively. Of the three bifunctionalizedpolyacrylamides tested, Polymer 4 demonstrates the best surfacerobustness.

The orthogonal polyacrylamides prepared by the procedure described aboveare generally random copolymers. It may be desirable to separatedifferent functional parts of the polymer architecture, for example,separating all the azide functional groups from all the amine functionalgroups to different segments of the polymer chain. This alternativesynthesis is readily achievable using controlled radical polymerization(CRP) methods (e.g., RAFT, ATRP). Scheme 2.1 and 2.2 demonstrate twosynthetic routes for preparing a block copolymer AEMA-b-AzAPA (Polymer7).

The coating performance of a block copolymer AEMA-b-AzAPA prepared by aRAFT technique according to Scheme 2.2 was compared to that of a randomcopolymer Polymer 4 on epoxy silane monolayer surface. The CVD processwas performed on the flow cell using a desiccator in an oven at 60° C.and the flow cell was incubated overnight. The flow cell layout issummarized in Table 6. The coupling reaction between the aminofunctional groups of Polymer 7 and Polymer 4 with the epoxy surface wasperformed at 60° C. for an hour.

TABLE 6 Polymer/ Epoxy Polymer surface Vol. of cou- cou- polymer plingpling used for Approx. [P5/ temp. time coating [polymer]/ P7]/ Channel(° C.) (min) (μL) Polymer w/v % μM 1 60 60 450 Polymer 7 0.3* 10 2 60 60450 Polymer 7 0.3* 10 3 60 60 450 Polymer 7 0.3* 10 4 60 60 450 Polymer7 0.3* 10 5 60 60 450 Polymer 4 0.5 10 6 60 60 450 Polymer 4 0.5 10 7 6060 450 Polymer 4 0.5 10 8 60 60 450 Polymer 4 0.5 10 *The solids contentof this batch, as measured by RI, was very high (4.9% Brix @ 0.3% w/v)

Two QC metrics (QC1 and QC3) were used to measure the success of themethod. The Typhoon florescence image of the polymers coated flow celland the related chart of median Typhoon intensity of the polymers on theepoxy silane monolayer surface for TET QC1 and TET QC3 are illustratedin FIGS. 4A, 4B, 4C and 4D respectively. The results of TET QC signalchange are shown in FIG. 4E. TET QC measurements for the epoxy surfaceare summarized in Table 7 below. Both materials yielded stable surfacesas measured by TET QC performed after a thermal Stress Test. In eachcase, the coatings were very uniform.

TABLE 7 % Intensity change, % Surface Lanes Polymer QC1->QC3 Loss 1Polymer 7 4% −4% 2 Polymer 7 4% −4% 3 Polymer 7 3% −3% 4 Polymer 7 2%−2% 5 Polymer 4 12% −12% 6 Polymer 4 13% −13% 7 Polymer 4 13% −13% 8Polymer 4 14% −14%

Example 2

Scheme 3 illustrates a flow chart of substrate preparation byimmobilizing a DNA copolymer to a silanized substrate surface. First,DNA copolymer is formed by reacting alkyne functionalized primers withacrylamide, azido-acrylamide and amino-acrylamide monomers to form apre-grafted ternary copolymer (DNA copolymer). The substrate surface isfirst treated with a silane comprising epoxy groups. Then the DNAcopolymer is immobilized to the substrate surface by reacting theprimary amino groups of the polymer with the epoxy groups of the silane.The architecture of the DNA copolymer prepared by this process may bemodified by addition of other monomers, for example,N,N-methylenebisacrylamide can be added to introduce crosslinking in adefined manner, or inimers (or monomer-initiators) can be added tointroduce branching points in a defined manner. Controlledpolymerization techniques such as RAFT, ATRP, or NMP may also be used tocreate block copolymer structures separating out the functional parts ofthe polymer to be more effective, if needed.

Example 3

Scheme 4 illustrates the reaction between bicyclo[6.1.0] non-4-yne(“BCN”) functionalized P5 or P7 primers with tetrazine modifiedacrylamide polymer (“Tz-Am”) to form a grafted polymer. Thiscatalyst-free, strain promoted click reaction can be performed at roomtemperature and it is compatible with aqueous environment. The resultinggrafted polymer can be purified using a number of methods, e.g.precipitation or tangential flow filtration (“TFF”) etc. Othernon-limiting possible polymer backbones that can be used in this processinclude polyacrylates or polyphosphazenes.

Scheme 5 illustrates the attachment of the pre-grafted tetrazineacrylamide polymer to norbornene functionalized surface of a substrate.The norbornene silanized surface is a standard part of the NextSeq®platform of Illumina. Alternatively, tetrazine functionalized polymerand BCN primers may be attached to the substrate surface in situ insteadof forming the grafted polymer.

To assess the feasibility of this approach, initial experiments werecarried out using a model system in a small scale solution reaction(Scheme 6).

Scheme 6 demonstrates the reaction between norbornene (Nb) and acommercially available bipyridyl tetrazine (BiPy) at 1:1 mole ratio. Thereaction was carried out at room temperature in an NMR tube, using CDCl₃as solvent with mild agitation. A NMR spectrum of the reaction mixturewas taken at three different time points, one at the beginning of thereaction (t=0), one at 15 minutes and one at 60 minutes. The NMR spectrashowed that the peak of the two alkene hydrogens of norbornene (withchemical shift at about 5.8 ppm) was disappearing and became almostinvisible after one hour (See FIG. 5). This indicates the rapid kineticsof the reaction between tetrazine and norbornene.

In a separate experiment, Scheme 7 demonstrates a facile strain promoted[4+2] cycloaddition of cyclooctyne (10 mM) with a bisphenyl substituted1,2,4,5-tetrazine (1 mM). The reaction was carried out at roomtemperature in dried MeOH. FIG. 6 shows the pattern of UV-vis absorptiondecrease of cyclooctyne which indicates the reaction was nearlycompleted after only 9 minutes. See W. Chen, D. Wang, C. Dai, D.Hamelberg B. Wang, Chem. Commun., 2012, 48, 1736-1738.

Example 4

Scheme 8 illustrates the preparation of a pre-grafted poly(glycidylmethacrylate) comb polymer by reacting the glycidyl ether groups of thepoly(glycidyl methacrylate) with the amino groups of the functionalizedprimers and amino-PEG-azide. This grafted polymer can be attached to astandard norbornene surface via catalyst-free, strain promoted clickreaction between the side chain azido groups of the polymer and thenorbornenes. A number of commercially available amino azides can be usedand the azido groups may also be replaced with other orthogonalfunctional groups.

Example 5

Scheme 9 illustrates the preparation of a pre-grafted poly(glycidylmethacrylate) comb polymer by reacting the glycidyl groups of thepoly(glycidyl methacrylate) with the amino groups of the functionalizedprimers and amino-PEG-Boc-amide. This grafted polymer is then subject toBoc-deprotection to generate the primary amino functionalized sidechain, which be attached to a glycidyl or epoxy functionalized surface.

Example 6

FIG. 7 illustrates the possible surface chemistry of a pre-grafteddendrimer with oligonucleotides bonded external surface. The originpoint of the dendrimer can be functionalized with an azido group fordirect surface attachment. Alternatively, the azido group can react withan alkyne group in the center point of a second dendrimer, wherein thesecond dendrimer has substrate attachment groups “A” covered externalsurface to create a Janus type particle for self-assembly.

Example 7

Orthogonal polymers with polyphosphazene backbone can also be used inthe present application. Polyphosphazenes can serve as linear scaffoldsfor possible branching of the polymer architecture, building dendronizedpolymers, or for subsequent polymer attachment. Scheme 10.1 illustratesa synthetic route utilizing the cyclic hexachlorophosphazene core forthe construction of modified acrylamide monomers.

Scheme 10.2 and 10.3 demonstrate the synthesis of two polyphosphazenescaffolds for subsequent polymer attachment. Several polyphosphazenesyntheses have been reported by Qiu et al., Nanotechnology, 18 (2007)475-602 and Cheng et al., Journal of Polymer Science, Part A: PolymerChemistry, 2013, 51, 1205-1214.

Scheme 10.4 illustrates two possible routes to prepare linearpolydichlorophosphazene (PDCP) backbone. Route 1 is the anioniccontrolled polymerization. Route 2 is the ring opening reaction ofhexachlorophosphazene. Route 1 is preferred with potential access tolinear, cyclo-linear and cross-linked polymer architecture, as well asthe possibility to introduce cross-linking.

What is claimed is:
 1. A substrate having a first surface comprising apolymer covalently bonded thereto, wherein the polymer comprises arecurring unit of Formula (IV):

wherein each of R^(1c) and R^(2c) is independently hydrogen, optionallysubstituted alkyl, or optionally substituted phenyl; R^(3c) is hydrogen,optionally substituted alkyl, optionally substituted phenyl, oroptionally substituted C₇₋₁₄ aralkyl; Ar is an optionally substitutedC₆₋₁₀ aryl or an optionally substituted 5 or 6 membered heteroaryl;R^(A) is optionally substituted tetrazine; and L³ is a single bond, anoptionally substituted alkylene linker, or an optionally substitutedheteroalkylene linker.
 2. The substrate of claim 1, wherein R^(1c) ishydrogen or optionally substituted alkyl and each R^(2c) and R^(3c) ishydrogen.
 3. The substrate of claim 1, wherein Ar is an optionallysubstituted phenyl.
 4. The substrate of claim 1, wherein the recurringunit of Formula (IV) is also represented by Formula (IVa):

wherein R^(1c) is selected from hydrogen or methyl.
 5. The substrate ofclaim 1, further comprises one or more recurring units selected from thegroup consisting of polyacrylamides, polyacrylates, polyurethanes,polysiloxanes, silicones, polyacroleins, polyphosphazenes,polyisocyanates, poly-ols, and polysaccharides, and combinationsthereof.
 6. The substrate of claim 5, further comprises one or morerecurring units of Formula (IIIa) or (IIIb) or both:

wherein each R^(4a), R^(4b) and R^(5b) is independently hydrogen or C₁₋₃alkyl; and each R^(5a), R^(6a), R^(6b) and R^(7b) is independentlyhydrogen, optionally substituted C₁₋₆ alkyl, or optionally substitutedphenyl.
 7. The substrate of claim 6, wherein the recurring unit ofFormula (IIIa) is also represented by (IIIa1), (IIIa2) or (IIIa3):


8. The substrate of claim 6, wherein the recurring unit of Formula(IIIb) is also represented by (IIIb1):


9. The substrate of claim 8, wherein the first surface of the substratecomprises functionalized silane comprising unsaturated moieties selectedfrom the group consisting of cycloalkenes, cycloalkynes,heterocycloalkenes, and heterocycloalkynes, and combinations thererof.10. The substrate of claim 9, wherein the covalent bonding between thepolymer and the first surface of the substrate comprises the structuremoiety

or combinations thereof, and wherein * indicates the polymer's point ofconnection with the first surface.
 11. The substrate of claim 9, furthercomprising functionalized oligonucleotides covalently bonded to thepolymer.
 12. The substrate of claim 11, wherein the polymer comprisesmodified recurring units of the structure:

wherein the covalent bonding position with the functionalized nucleotideis shown by a bond with a wavy line.
 13. The substrate of claim 11,wherein the functionalized oligonucleotides are covalently bonded to thepolymer through reaction of one or more functional moieties on thefunctionalized oligonucleotides with optionally substituted tetrazine,and wherein the functional moieties comprises alkynes, cycloalkenes,cycloalkynes, heterocycloalkenes, heterocycloalkynes, or optionallysubstituted variants or combinations thereof.
 14. The substrate of claim13, wherein the functional moieties comprise alkynes or optionallysubstituted variants thereof.
 15. The substrate of claim 1, comprisingboth polymer coated regions and inert regions.
 16. The substrate ofclaim 15, wherein the polymer-coated regions are wells and the inertregions are interstitial regions.
 17. A polymer for surfacefunctionalization, wherein the polymer comprises a recurring unit ofFormula (IV):

wherein each of R^(1c) and R^(2c) is independently hydrogen, optionallysubstituted alkyl, or optionally substituted phenyl; R^(3c) is hydrogen,optionally substituted alkyl, optionally substituted phenyl, oroptionally substituted C₇₋₁₄ aralkyl; Ar is an optionally substitutedC₆₋₁₀ aryl or an optionally substituted 5 or 6 membered heteroaryl;R^(A) is optionally substituted tetrazine; and L³ is a single bond, anoptionally substituted alkylene linker, or an optionally substitutedheteroalkylene linker.
 18. A method for immobilizing a polymer to afirst surface of a substrate, comprising: contacting a polymercomprising a recurring unit of Formula (IV) with the first surface ofthe substrate, said first surface comprising a first plurality offunctional groups covalently attached thereto; and reacting the firstplurality of functional groups of the first surface with the polymer,thereby covalently bonding the polymer to the first surface of thesubstrate;

wherein each of R^(1c) and R^(2c) is independently hydrogen, optionallysubstituted alkyl, or optionally substituted phenyl; R^(3c) is hydrogen,optionally substituted alkyl, optionally substituted phenyl, oroptionally substituted C₇₋₁₄ aralkyl; Ar is an optionally substitutedC₆₋₁₀ aryl or an optionally substituted 5 or 6 membered heteroaryl;R^(A) is optionally substituted tetrazine; and L³ is a single bond, anoptionally substituted alkylene linker, or an optionally substitutedheteroalkylene linker.
 19. The method of claim 18, wherein the firstplurality of functional groups of the first surface comprise vinyl,acryloyl, alkenyl, cycloalkenyl, heterocycloalkenyl, alkynyl,cycloalkynyl, heterocycloalkynyl, nitrene, aldehyde, hydrazinyl,glycidyl ether, epoxy, carbene, isocyanate or maleimide, or optionallysubstituted variants, or combinations thereof.
 20. The method of claim18, wherein the first plurality of functional groups comprise norbornenegroups.